Predicting t cell epitopes useful for vaccination

ABSTRACT

The present invention relates to methods for predicting T cell epitopes useful for vaccination. In particular, the present invention relates to methods for predicting whether modifications in peptides or polypeptides such as tumor-associated neoantigens are immunogenic and, in particular, useful for vaccination, or for predicting which of such modifications are most immunogenic and, in particular, most useful for vaccination. The methods of the invention may be used, in particular, for the provision of vaccines which are specific for a patient&#39;s tumor and thus, in the context of personalized cancer vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase patent/application ofPCT/EP2016/052684, filed on Feb. 9, 2016. which claims priority toInternational Patent Application No. PCT/EP2015/053021, filed on Feb.12, 2015, the disclosures of each of which are hereby incorporated byreference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392041500SeqList.txt.date recorded: Aug. 10, 2017, size: 33 KB).

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods for predicting T cell epitopesuseful for vaccination. In particular, the present invention relates tomethods for predicting whether modifications in peptides or polypeptidessuch as tumor-associated neoantigens are immunogenic and, in particular,useful for vaccination, or for predicting which of such modificationsarc most immunogenic and, in particular, most useful for vaccination.The methods of the invention may be used, in particular, for theprovision of vaccines which are specific for a patient's tumor and,thus, in the context of personalized cancer vaccines.

BACKGROUND OF THE INVENTION

Mutations are regarded as ideal targets for cancer immunotherapy. Asneo-epitopes with strict lack of expression in any healthy tissue, theyare expected to be safe and could bypass the central tolerancemechanisms. The systematic use of mutations for vaccine approaches,however, is hampered by the uniqueness of the repertoire of mutations(“the mutanome”) in every patient's tumor (Alexandrov. L. B., et al.,Nature 500, 415 (2013)). We have recently proposed a personalizedimmunotherapy approach targeting the spectrum of individual mutations(Castle, J. C., et al., Cancer Res 72, 1081 (2012)).

However, (here is a need for a model to predict whether an epitope, inparticular a neo-epitope, will induce efficient immunity and, thus, willbe useful in vaccination.

Here we show in three independent murine tumor models that aconsiderable fraction of non-synonymous cancer mutations is immunogenicand that unexpectedly the immunogenic mutanome is pre-dominantlyrecognized by CD4⁺ T cells (“the CD4+ immunome”). Vaccination with suchCD4⁺ immunogenic mutations confers strong anti-tumour activity.Encouraged by these findings we set up a process comprising mutationdetection by exome sequencing, selection of vaccine targets by solelybioinformatical prioritization of mutated epitopes predicted to beabundantly expressed and good MHC class II binders and rapid productionof synthetic mRNA vaccines encoding multiple of these mutated epitopes.We show that vaccination with such poly-neo-epitopic mRNA vaccinesinduces potent tumor control and complete rejection of establishedaggressively growing tumors in mice. Moreover, we demonstrate that CD4⁺T cell neo-epitope vaccination induces CTL responses against anindependent immunodominant antigen in tumor bearing mice indicatingorchestration of antigen spread. Finally, we demonstrate by analyses ofcorresponding human cancer types with the same bioinformaticalalgorithms the abundance of mutations predicted to bind to MHC class IIin human cancers as well. Thus, the tailored immunotherapy approachintroduced here may be regarded as a universally applicable blueprintfor comprehensive exploitation of the huge neo-epitope target repertoireof cancers enabling targeting of even patient's tumour with “just intime” produced vaccines.

DESCRIPTION OF INVENTION Summary of the Invention

In one aspect, the present invention relates to a method for predictingimmunogenic amino acid modifications, the method comprising the steps:

a) ascertaining a score for binding of a modified peptide which is afragment of a modified protein to one or more MHC class II molecules.and

b) ascertaining a score for expression or abundance of the modifiedprotein.

In one embodiment, a score for binding to one or more MHC class IImolecules indicating binding to one or more MHC class II molecules and ascore for expression or abundance of the modified protein indicatingexpression, high level of expression or abundance of the modifiedprotein indicates that the modification or modified peptide isimmunogenic. In a further aspect, the present invention relates to amethod for selecting and/or ranking immunogenic amino acidmodifications, the method comprising the steps:

a) ascertaining a score for binding of a modified peptide which is afragment of a modified protein to one or more MHC class II molecules,and

b) ascertaining a score for expression or abundance of the modifiedprotein,

wherein the method comprises performing steps a) and b) on two or moredifferent modifications.

In one embodiment, the different modifications are present in the sameand/or in different proteins.

In one embodiment, the method comprises comparing the scores of said twoor more different modifications. In one embodiment, the scores of saidtwo or more different modifications are compared by ranking thedifferent modifications by their MHC class II binding scores andremoving modifications with an expression or abundance of less than agiven threshold.

In one embodiment of all aspects of the invention, the score for bindingto one or more MHC class II molecules reflects a probability for bindingto one or more MHC class II molecules. In one embodiment, the score forbinding to one or more MHC class II molecules is ascertained by aprocess comprising a sequence comparison with a database of MHC classII-binding motifs.

In one embodiment of all aspects of the invention, the method comprisesperforming step a) on two or more different modified peptides, said twoor more different modified peptides comprising the same modification(s).In one embodiment, the two or more different modified peptidescomprising the same modification(s) comprise different fragments of amodified protein, said different fragments comprising the samemodification(s) present in the protein. In one embodiment, the two ormore different modified peptides comprising the same modification(s)comprise different potential MHC class II binding fragments of amodified protein, said fragments comprising the same modification(s)present in the protein. In one embodiment, the method further comprisesselecting (the) modified peptide(s) from the two or more differentmodified peptides comprising the same modification(s) having aprobability or having the highest probability for binding to one or moreMHC class II molecules. In one embodiment, the two or more differentmodified peptides comprising the same modification(s) differ in lengthand/or position of the modification(s). In one embodiment, the bestscore for binding to one or more MHC class II molecules of the two ormore different modified peptides comprising the same modification(s) isassigned to the modification(s).

In one embodiment of all aspects of the invention, ascertaining a scorefor expression or abundance of a modified protein comprises determiningthe level of expression of the protein to which the modification isassociated and determining the frequency of the modified protein amongthe protein to which the modification is associated. In one embodiment,said determining the level of expression of the protein to which themodification is associated and/or determining the frequency of themodified protein among the protein to which the modification isassociated is performed on the RNA level. In one embodiment, thefrequency of the modified protein among the protein to which themodification is associated is determined by determining the variantallele frequency. In one embodiment, the variant allele frequency is thesum of detected sequences, in particular reads, covering the mutationsite and carrying the mutation divided by the sum of all detectedsequences, in particular reads, covering the mutation site. In oneembodiment, the variant allele frequency is the sum of mutatednucleotides at the mutation site divided by the sum of all nucleotidesdetermined at the mutation site. In one embodiment, for ascertaining ascore for expression or abundance of a modified protein a score for thelevel of expression of the protein to which the modification isassociated is multiplied with a score for the frequency of the modifiedprotein among the protein lo which the modification is associated.

In one embodiment of all aspects of the invention, the modified peptidecomprises a fragment of the modified protein, said fragment comprisingthe modification present in the protein.

In one embodiment of all aspects of the invention, the method furthercomprises identifying non-synonymous mutations in one or moreprotein-coding regions.

In one embodiment of all aspects of the invention, amino acidmodifications are identified by partially or completely sequencing thegenome or transcriptome of one or more cells such as one or more cancercells and optionally one or more non-cancerous cells and identifyingmutations in one or more protein-coding regions. In one embodiment, saidmutations arc somatic mutations. In one embodiment, said mutations arecancer mutations.

In one embodiment of all aspects of the invention, the method is used inthe manufacture of a vaccine. In one embodiment, the vaccine is derivedfrom (a) modification(s) or (a) modified peptide(s) predicted asimmunogenic or more immunogenic by said method.

In one embodiment, in particular in order to provide a personalizedvaccine for a patient such as a cancer patient, the modification(s) arepresent in said patient and said ascertaining a score for binding to oneor more MHC class II molecules, and said ascertaining a score forexpression or abundance of the modified protein is performed for saidpatient. Preferably, said one or more MHC class II molecules arc presentin said patient (in this embodiment the present invention may includedetermining the partial or complete MHC class II expression pattern ofthe patient). Preferably, said ascertaining a score for expression orabundance of the modified protein is performed on a sample from saidpatient such as a tumor specimen.

In a further aspect, the present invention relates to a method forproviding a vaccine comprising the step:

identifying (a) modification(s) or (a) modified peptide(s) predicted asimmunogenic or more immunogenic (than other modification(s) or modifiedpeptide(s) also analysed) by the method of the invention. In oneembodiment, the method further comprises the step:

providing a vaccine comprising a peptide or polypeptide comprising themodification(s) or modified peptide(s) predicted as immunogenic or moreimmunogenic, or a nucleic acid encoding the peptide or polypeptide.

In a further aspect, the present invention provides a vaccine which isobtainable using the methods according to the invention. Preferredembodiments of such vaccines arc described herein.

A vaccine provided according to the invention may comprise apharmaceutically acceptable carrier and may optionally comprise one ormore adjuvants, stabilizers etc. The vaccine may in the form of atherapeutic or prophylactic vaccine.

Another aspect relates to a method for inducing an immune response in apatient, comprising administering to the patient a vaccine providedaccording to the invention.

Another aspect relates lo a method of treating a cancer patientcomprising the steps:

(a) providing a vaccine using the methods according to the invention;and

(b) administering said vaccine to the patient.

Another aspect relates to a method of treating a cancer patientcomprising administering the vaccine according to the invention to thepatient.

In further aspects, the invention provides the vaccines described hereinfor use in the methods of treatment described herein, in particular foruse in treating or preventing cancer.

The treatments of cancer described herein can be combined with surgicalresection and/or radiation and/or traditional chemotherapy.

Other features and advantages of the instant invention will he apparentfrom the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”. H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds.,(1995) Helvetica ChimicaActa, CH-4010 Basel, Switzerland.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of biochemistry, cell biology,immunology, and recombinant DNA techniques which are explained in theliterature in the field (cf., e.g., Molecular Cloning: A LaboratoryManual, 2^(nd) Edition, J. Sambrook et al. eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a staled member, integer or step or group of members, integers orsteps but not the exclusion of any other member, integer or step orgroup of members, integers or steps although in some embodiments suchother member, integer or step or group of members, integers or steps maybe excluded, i.e. the subject-matter consists in the inclusion of astated member, integer or step or group of members, integers or steps.The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”), provided herein is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed. No language in the specification should be construedas indicating any non-claimed element essential to the practice of theinvention.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

According to the present invention, the term “peptide” refers tosubstances comprising two or more, preferably 3 or more, preferably 4 ormore, preferably 6 or more, preferably 8 or more, preferably 10 or more,preferably 13 or more, preferably 16 more, preferably 21 or more and upto preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acidsjoined covalently by peptide bonds. The term “polypeptide” or “protein”refers to large peptides, preferably to peptides with more titan 100amino acid residues, but in general the terms “peptide”, “polypeptide”and “protein” are synonyms and are used interchangeably herein.

According to the invention, the term “modification” with respect topeptides, polypeptides or proteins relates to a sequence change in apeptide, polypeptide or protein compared to a parental sequence such asthe sequence of a wildtype peptide, polypeptide or protein. The termincludes amino acid insertion variants, amino acid addition variants,amino acid deletion variants and amino acid substitution variants,preferably amino acid substitution variants. All these sequence changesaccording to the invention may potentially create new epitopes.

Amino acid insertion variants comprise insertions of single or two ormore amino acids in a particular amino acid sequence.

Amino acid addition variants comprise amino- and/or carboxy-terminalfusions of one or more amino acids, such as 1, 2, 3, 4 or 5, or moreamino acids.

Amino acid deletion variants are characterized by the removal of one ormore amino acids from the sequence, such as by removal of 1, 2, 3, 4 or5, or more amino acids.

Amino acid substitution variants arc characterized by at least oneresidue in the sequence being removed and another residue being insertedin its place.

According to the invention, a modification or modified peptide used fortesting in the methods of the invention may be derived from a proteincomprising a modification.

The term “derived” means according to the invention that a particularentity, in particular a particular peptide sequence, is present in theobject from which it is derived. In the case of amino acid sequences,especially particular sequence regions, “derived” in particular meansthat the relevant amino acid sequence is derived from an amino acidsequence in which it is present.

A protein comprising a modification from which a modification ormodified peptide used for testing in the methods of the invention may bederived may be a neoantigen.

According to the invention, the term “neoantigen” relates to a peptideor protein including one or more amino acid modifications compared tothe parental peptide or protein. For example, the neoantigen may be atumor-associated neoantigen, wherein the term “tumor-associatedneoantigen” includes a peptide or protein including amino acidmodifications due to tumor-specific mutations.

According to the invention, the term “tumor-specific mutation” or“cancer-specific mutation” relates to a somatic mutation that is presentin the nucleic acid of a tumor or cancer cell but absent in the nucleicacid of a corresponding normal, i.e. non-tumorous or non-cancerous,cell. The terms “tumor-specific mutation” and “tumor mutation” and theterms “cancer-specific mutation” and “cancer mutation” are usedinterchangeably herein.

The term “immune response” refers to an integrated bodily response to atarget such as an antigen and preferably refers to a cellular immuneresponse or a cellular as well as a humoral immune response. The immuneresponse may be protective/preventive/prophylactic and/or therapeutic.

“Inducing an immune response” may mean that there was no immune responsebefore induction, but it may also mean that there was a certain level ofimmune response before induction and after induction said immuneresponse is enhanced. Thus, “inducing an immune response” also includes“enhancing an immune response”. Preferably, after inducing an immuneresponse in a subject, said subject is protected from developing adisease such as a cancer disease or the disease condition is amelioratedby inducing an immune response. For example, an immune response againsta tumor-expressed antigen may be induced in a patient having a cancerdisease or in a subject being at risk of developing a cancer disease.Inducing an immune response in this case may mean that the diseasecondition of the subject is ameliorated, that the subject does notdevelop metastases, or that the subject being at risk of developing acancer disease does not develop a cancer disease.

The terms “cellular immune response” and “cellular response” or similarterms refer to an immune response directed to cells characterized bypresentation of an antigen with class I or class II MHC involving Tcells or T-lymphocytes which act as either “helpers” or “killers”. Thehelper T cells (also termed CD4⁺ T cells) play a central role byregulating the immune response and the killer cells (also termedcytotoxic T cells, cytolytic T cells, CD8⁺ T cells or CTLs) killdiseased cells such as cancer cells, preventing the production of morediseased cells. In preferred embodiments, The present invention involvesthe stimulation of an anti-tumor CTL response against tumor cellsexpressing one or more tumor-expressed antigens and preferablypresenting such tumor-expressed antigens with class I MHC.

An “antigen” according to the invention covers any substance, preferablya peptide or protein, that is a target of and/or induces an immuneresponse such as a specific reaction with antibodies or T-lymphocytes (Tcells). Preferably, an antigen comprises at least one epitope such as aT cell epitope. Preferably, an antigen in the context of the presentinvention is a molecule which, optionally after processing, induces animmune reaction, which is preferably specific for the antigen (includingcells expressing the antigen). The antigen or a T cell epitope thereofis preferably presented by a cell, preferably by an antigen presentingcell which includes a diseased cell, in particular a cancer cell, in thecontext of MHC molecules, which results in an immune response againstthe antigen (including cells expressing the antigen).

In one embodiment, an antigen is a tumor antigen (also termedtumor-expressed antigen herein), i.e., a part of a tumor cell such as aprotein or peptide expressed in a tumor cell which may be derived fromthe cytoplasm, the cell surface or the cell nucleus, in particular thosewhich primarily occur intracellularly or as surface antigens of tumorcells. For example, tumor antigens include the carcinoembryonal antigen,α1-fetoprotein, isoferritin, and fetal sulphoglycoprotein,α2-H-ferroprotein and γ-fetoprotein. According to the present invention,a tumor antigen preferably comprises any antigen which is expressed inand optionally characteristic with respect to type and/or expressionlevel for tumors or cancers as well as for tumor or cancer cells, i.e. atumor-associated antigen. In one embodiment, the term “tumor-associatedantigen” relates to proteins that are under normal conditionsspecifically expressed in a limited number of tissues and/or organs orin specific developmental stages, for example, the tumor-associatedantigens may be under normal conditions specifically expressed instomach tissue, preferably in the gastric mucosa, in reproductiveorgans, e.g., in testis, in trophoblastic tissue, e.g., in placenta, orin germ line cells, and are expressed or aberrantly expressed in one ormore tumor or cancer tissues. In this context, “a limited number”preferably means not more than 3, more preferably not more than 2. Thetumor antigens in the context of the present invention include, forexample, differentiation antigens, preferably cell type specificdifferentiation antigens, i.e., proteins that are under normalconditions specifically expressed in a certain cell type at a certaindifferentiation stage, cancer/testis antigens, i.e., proteins that areunder normal conditions specifically expressed in testis and sometimesin placenta, and germ line specific antigens. Preferably, the tumorantigen or the aberrant expression of the tumor antigen identifiescancer cells. In the context of the present invention, the tumor antigenthat is expressed by a cancer cell in a subject, e.g., a patientsuffering from a cancer disease, is preferably a self-protein in saidsubject. In preferred embodiments, the tumor antigen in the context ofthe present invention is expressed under normal conditions specificallyin a tissue or organ that is non-essential, i.e., tissues or organswhich when damaged by the immune system do not lead to death of thesubject, or in organs or structures of the body which are not or onlyhardly accessible by the immune system.

According to the invention, the terms “tumor antigen”, “tumor-expressedantigen”, “cancer antigen” and “cancer-expressed antigen” areequivalents and are used interchangeably herein.

The term “immunogenicity” relates to the relative effectivity to inducean immune response that is preferably associated with therapeutictreatments, such as treatments against cancers. As used herein, the term“immunogenic” relates to the property of having immunogenicity. Forexample, the term “immunogenic modification” when used in the context ofa peptide, polypeptide or protein relates to the effectivity of saidpeptide, polypeptide or protein to induce an immune response that iscaused by and/or directed against said modification. Preferably, thenon-modified peptide, polypeptide or protein docs not induce an immuneresponse, induces a different immune response or induces a differentlevel, preferably a lower level, of immune response.

According to the invention, the term “immunogenicity” or “immunogenic”preferably relates to the relative effectively to induce a biologicallyrelevant immune response, in particular an immune response which isuseful for vaccination. Thus, in one preferred embodiment, an amino acidmodification or modified peptide is immunogenic if it induces an immuneresponse against the target modification in a subject, which immuneresponse may be beneficial for therapeutic or prophylactic purposes.

The terms “major histocompatibility complex” and the abbreviation “MHC”include MHC class I and MHC class II molecules and relate to a complexof genes which occurs in all vertebrates. MHC proteins or molecules areimportant for signaling between lymphocytes and antigen presenting cellsor diseased cells in immune reactions, wherein the MHC proteins ormolecules bind peptides and present them for recognition by T cellreceptors. The proteins encoded by the MHC are expressed on the surfaceof cells, and display both self antigens (peptide fragments from thecell itself) and non-self antigens (e.g., fragments of invadingmicroorganisms) to a T cell.

The MHC region is divided into three subgroups, class I, class II, andclass III. MHC class I proteins contain an α-chain and β2-microglobulin(not part of the MHC encoded by chromosome 15). They present antigenfragments to cytotoxic T cells. On most immune system cells,specifically on antigen-presenting cells, MHC class II proteins containα- and β-chains and they present antigen fragments to T-helper cells.MHC class III region encodes for other immune components, such ascomplement components and some that encode cytokines.

The MHC is both polygenic (there are several MHC class I and MHC classII genes) and polymorphic (there are multiple alleles of each gene).

As used herein, the term “haplotype” refers to the HLA alleles found onone chromosome and the proteins encoded thereby. Haplotype may alsorefer to the allele present at any one locus within the MHC. Each classof MHC is represented by several loci: e.g., HLA-A (Human LeukocyteAntigen-A), HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K,HLA-L, HLA-P and HLA-V for class I and HLA-DRA, HLA-DRB1-9, HLA-,HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DMA, HLA-DMB, HLA-DOA, andHLA-DOB for class II. The terms “HLA allele” and “MHC allele” are usedinterchangeably herein.

The MHCs exhibit extreme polymorphism: within the human population thereare, at each genetic locus, a great number of haplotypes comprisingdistinct alleles. Different polymorphic MHC alleles, of both class I andclass II, have different peptide specificities: each allele encodesproteins that bind peptides exhibiting particular sequence patterns.

In one preferred embodiment of all aspects of the invention an MHCmolecule is an HLA molecule.

According to the invention, MHC class II includes HLA-DM, HLA-DO,HLA-DP, HLA-DQ and HLA-DR.

In the context of the present invention, the term “MHC binding peptide”includes MHC class I and/or class II binding peptides or peptides thatcan be processed to produce MHC class I and/or class II bindingpeptides. In the case of class I MHC/peptide complexes, the bindingpeptides are typically 8-12, preferably 8-10 amino acids long althoughlonger or shorter peptides may be effective. In the case of class IIMHC/peptide complexes, the binding peptides are typically 9-30,preferably 10-25 amino acids long and are in particular 13-18 aminoacids long, whereas longer and shorter peptides may be effective.

If a peptide is to be presented directly, i.e., without processing, inparticular without cleavage, it has a length which is suitable forbinding to an MHC molecule, in particular a class I MHC molecule, andpreferably is 7-30 amino acids in length such as 7-20 amino acids inlength, more preferably 7-12 amino acids in length, more preferably 8-11amino acids in length, in particular 9 or 10 amino acids in length.

If a peptide is part of a larger entity comprising additional sequences,e.g. of a vaccine sequence or polypeptide, and is to be presentedfollowing processing, in particular following cleavage, the peptideproduced by processing has a length which is suitable for binding to anMHC molecule, in particular a class I MHC molecule, and preferably is7-30 amino acids in length such as 7-20 amino acids in length, morepreferably 7-12 amino acids in length, more preferably 8-11 amino acidsin length, in particular 9 or 10 amino acids in length. Preferably, thesequence of the peptide which is to be presented following processing isderived from the amino acid sequence of an antigen or polypeptide usedfor vaccination, i.e., its sequence substantially corresponds and ispreferably completely identical to a fragment of the antigen orpolypeptide.

Thus, an MHC binding peptide in one embodiment comprises a sequencewhich substantially corresponds and is preferably completely identicalto a fragment of an antigen.

The term “epitope” refers to an antigenic determinant in a molecule suchas an antigen, i.e., to a part in or fragment of the molecule that isrecognized by the immune system, for example, that is recognized by a Tcell, in particular when presented in the context of MHC molecules. Anepitope of a protein such as a tumor antigen preferably comprises acontinuous or discontinuous portion of said protein and is preferablybetween 5 and 100, preferably between 5 and 50, more preferably between8 and 30, most preferably between 10 and 25 amino acids in length, forexample, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. It isparticularly preferred that the epitope in the context of the presentinvention is a T cell epitope.

According to the invention an epitope may bind to MHC molecules such asMHC molecules on the surface of a cell and thus, may be a “MHC bindingpeptide”.

As used herein the term “neo-epitope” refers to an epitope that is notpresent in a reference such as a normal non-cancerous or germline cellbut is found in cancer cells. This includes, in particular, situationswherein in a normal non-cancerous or germline cell a correspondingepitope is found, however, due to one or more mutations in a cancer cellthe sequence of the epitope is changed so as to result in theneo-epitope.

As used herein, the term “T cell epitope” refers to a peptide whichhinds to a MHC molecule in a configuration recognized by a T cellreceptor. Typically, T cell epitopes are presented on the surface of anantigen-presenting cell.

As used herein, the term “predicting immunogenic amino acidmodifications” refers to a prediction whether a peptide comprising suchamino acid modification will be immunogenic and thus useful as epitope,in particular T cell epitope, in vaccination.

According to the invention, a T cell epitope may be present in a vaccineas a part of a larger entity such as a vaccine sequence and/or apolypeptide comprising more than one T cell epitope. The presentedpeptide or T cell epitope is produced following suitable processing.

T cell epitopes may be modified at one or more residues that are notessential for TCR recognition or for binding to MHC. Such modified Tcell epitopes may be considered immunologically equivalent.

Preferably a T cell epitope when presented by MHC and recognized by a Tcell receptor is able to induce in the presence of appropriateco-stimulatory signals, clonal expansion of the T cell carrying the Tcell receptor specifically recognizing the peptide/MHC-complex.

Preferably, a T cell epitope comprises an amino acid sequencesubstantially corresponding to the amino acid sequence of a fragment ofan antigen. Preferably, said fragment of an antigen is an MHC class Iand/or class II presented peptide.

A T cell epitope according to the invention preferably relates to aportion or fragment of an antigen which is capable of stimulating animmune response, preferably a cellular response against the antigen orcells characterized by expression of the antigen and preferably bypresentation of the antigen such as diseased cells, in particular cancercells. Preferably, a T cell epitope is capable of stimulating a cellularresponse against a cell characterized by presentation of an antigen withclass I MHC and preferably is capable of stimulating anantigen-responsive cytotoxic T-lymphocyte (CTL). “Antigen processing” or“processing” refers to the degradation of a peptide, polypeptide orprotein into procession products, which are fragments of said peptide,polypeptide or protein (e.g., the degradation of a polypeptide intopeptides) and the association of one or more of these fragments (e.g.,via binding) with MHC molecules for presentation by cells, preferablyantigen presenting cells, to specific T cells.

“Antigen presenting cells” (APC) are cells which present peptidefragments of protein antigens in association with MHC molecules on theircell surface. Some APCs may activate antigen specific T cells.

Professional antigen-presenting cells are very efficient atinternalizing antigen, either by phagocytosis or by receptor-mediatedendocytosis. and then displaying a fragment of the antigen, bound to aclass II MHC molecule, on their membrane. The T cell recognizes andinteracts with the antigen-class II MHC molecule complex on the membraneof the antigen-presenting cell. An additional co-stimulatory signal isthen produced by the antigen-presenting cell, leading to activation ofthe T cell. The expression of co-stimulatory molecules is a definingfeature of professional antigen-presenting cells.

The main types of professional antigen-presenting cells are dendriticcells, which have the broadest range of antigen presentation, and areprobably the most important antigen-presenting cells, macrophages,B-cells, and certain activated epithelial cells. Dendritic cells (DCs)are leukocyte populations that present antigens captured in peripheraltissues to T cells via both MHC class II and I antigen presentationpathways. It is well known that dendritic cells are potent inducers ofimmune responses and the activation of these cells is a critical stepfor the induction of antitumoral immunity. Dendritic cells areconveniently categorized as “immature” and “mature” cells, which can beused as a simple way to discriminate between two well characterizedphenotypes. However, this nomenclature should not be construed toexclude all possible intermediate stages of differentiation. Immaturedendritic cells are characterized as antigen presenting cells with ahigh capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g. CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86and 4-1 BB). Dendritic cell maturation is referred to as the status ofdendritic cell activation at which such antigen-presenting dendriticcells lead to T cell priming, while presentation by immature dendriticcells results in tolerance. Dendritic cell maturation is chiefly causedby biomolecules with microbial features detected by innate receptors(bacterial DNA, viral RNA, endotoxin, etc.). pro-inflammatory cytokines(TNF, IL-1, IFNs), ligation of CD40 on the dendritic cell surface byCD40L, and substances released from cells undergoing stressful celldeath. The dendritic cells can be derived by culturing bone marrow cellsin vitro with cytokines, such as granulocyte-macrophagecolony-stimulating factor (GM-CSF) and tumor necrosis factor alpha.

Non-professional antigen-presenting cells do not constitutively expressthe MHC class II proteins required for interaction with naive T cells;these are expressed only upon stimulation of the non-professionalantigen-presenting cells by certain cytokines such as IFNγ.

Antigen presenting cells can be loaded with MHC class I presentedpeptides by transducing the cells with nucleic acid, preferably RNA,encoding a peptide or polypeptide comprising the peptide to bepresented, e.g. a nucleic acid encoding an antigen or polypeptide usedfor vaccination.

In some embodiments, a pharmaceutical composition or vaccine comprisinga nucleic acid delivery vehicle that targets a dendritic or otherantigen presenting cell may be administered to a patient, resulting intransfection that occurs in vivo. In vivo transfection of dendriticcells, for example, may generally be performed using any methods knownin the art, such as those described in WO 97/24447, or the gene gunapproach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.

According to the invention, the term “antigen presenting cell” alsoincludes target cells.

“Target cell” shall mean a cell which is a target tor an immune responsesuch as a cellular immune response. Target cells include cells thatpresent an antigen, i.e. a peptide fragment derived from an antigen, andinclude any undesirable cell such as a cancer cell. In preferredembodiments, the target cell is a cell expressing an antigen asdescribed herein and preferably presenting said antigen with class IMHC.

The term “portion” refers lo a fraction. With respect to a particularstructure such as an amino acid sequence or protein the term “portion”thereof may designate a continuous or a discontinuous fraction of saidstructure. Preferably, a portion of an amino acid sequence comprises atleast 1%, at least 5%, at least 10%, at least 20%, at least 30%,preferably at least 40%, preferably at least 50%, more preferably atleast 60%, more preferably at least 70%, even more preferably at least80%, and most preferably at least 90% of the amino acids of said aminoacid sequence. Preferably, if the portion is a discontinuous fractionsaid discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or moreparts of a structure, each part being a continuous element of thestructure. For example, a discontinuous fraction of an amino acidsequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably notmore than 4 parts of said amino acid sequence, wherein each partpreferably comprises at least 5 continuous amino acids, at least 10continuous amino acids, preferably at least 20 continuous amino acids,preferably at least 30 continuous amino acids of the amino acidsequence.

The terms “part” and “fragment” are used interchangeably herein andrefer to a continuous clement. For example, a part of a structure suchas an amino acid sequence or protein refers to a continuous element ofsaid structure. A portion, a part or a fragment of a structurepreferably comprises one or more functional properties of saidstructure. For example, a portion, a part or a fragment of an epitope,peptide or protein is preferably immunologically equivalent to theepitope, peptide or protein it is derived from. In the context of thepresent invention, a “part” of a structure such as an amino acidsequence preferably comprises, preferably consists of at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, at least 99% of the entirestructure or amino acid sequence.

The term “immunoreactive cell” in the context of the present inventionrelates to a cell which exerts effector functions during an immunereaction. An “immunoreactive cell” preferably is capable of binding anantigen or a cell characterized by presentation of an antigen or apeptide fragment thereof (e.g. a T cell epitope) and mediating an immuneresponse. For example, such cells secrete cytokines and/or chemokines,secrete antibodies, recognize cancerous cells, and optionally eliminatesuch cells. For example, immunoreactive cells comprise T cells(cytotoxic T cells, helper T cells, tumor infiltrating T cells), Bcells, natural killer cells, neutrophils, macrophages, and dendriticcells. Preferably, in the context of the present invention,“immunoreactive cells” are T cells, preferably CD4⁺ and/or CD8⁺ T cells.

Preferably, an “immunoreactive cell” recognizes an antigen or a peptidefragment thereof with some degree of specificity, in particular ifpresented in the context of MHC molecules such as on the surface ofantigen presenting cells or diseased cells such as cancer cells.Preferably, said recognition enables the cell that recognizes an antigenor a peptide fragment thereof to be responsive or reactive. If the cellis a helper T cell (CD4⁺ T cell) bearing receptors that recognize anantigen or a peptide fragment thereof in the context of MHC class IImolecules such responsiveness or reactivity may involve the release ofcytokines and/or the activation of CD8⁺ lymphocytes (CTLs) and/orB-cells. If the cell is a CTL such responsiveness or reactivity mayinvolve the elimination of cells presented in the context of MHC class Imolecules, i.e., cells characterized by presentation of an antigen withclass I MHC, for example, via apoptosis or perform-mediated cell lysis.According to the invention, CTL responsiveness may include sustainedcalcium flux, cell division, production of cytokines such as IFN-γ andTNF-α, up-regulation of activation markers such as CD44 and CD69, andspecific cytolytic killing of antigen expressing target cells. CTLresponsiveness may also be determined using an artificial reporter thataccurately indicates CTL responsiveness. Such CTL that recognize anantigen or an antigen fragment and are responsive or reactive arc alsotermed “antigen-responsive CTL” herein. If the cell is a B cell suchresponsiveness may involve the release of immunoglobulins.

The terms “T cell” and “T lymphocyte” are used interchangeably hereinand include T helper cells (CD4⁺ T cells) and cytotoxic T cells (CTLs,CD8⁺ T cells) which comprise cytolytic T cells.

T cells belong to a group of white blood cells known as lymphocytes, andplay a central role in cell-mediated immunity. They can be distinguishedfrom other lymphocyte types, such as B cells and natural killer cells bythe presence of a special receptor on their cell surface called T cellreceptor (TCR). The thymus is the principal organ responsible for thematuration of T cells. Several different subsets of T cells have beendiscovered, each with a distinct function.

T helper cells assist other white blood cells in immunologic processes,including maturation of B cells into plasma cells and activation ofcytotoxic T cells and macrophages, among other functions. These cellsarc also known as CD4⁺ T cells because they express the CD4 protein ontheir surface. Helper T cells become activated when they are presentedwith peptide antigens by MHC class II molecules that are expressed onthe surface of antigen presenting cells (APCs). Once activated, theydivide rapidly and secrete small proteins called cytokines that regulateor assist in the active immune response.

Cytotoxic T cells destroy virally infected cells and tumor cells, andare also implicated in transplant rejection. These cells are also knownas CD8+ T cells since they express the CD8 glycoprotein at theirsurface. These cells recognize their targets by binding to antigenassociated with MHC class I, which is present on the surface of nearlyevery cell of the body.

A majority of T cells have a T cell receptor (TCR) existing as a complexof several proteins. The actual T cell receptor is composed of twoseparate peptide chains, which are produced from the independent T cellreceptor alpha and beta (TCRα and TCRβ) genes and are called α- andβ-TCR chains. γδ T cells (gamma delta T cells) represent a small subsetof T cells that possess a distinct T cell receptor (TCR) on theirsurface. However, in γδ T cells, the TCR is made up of one γ-chain andone δ-chain. This group of T cells is much less common (2% of total Tcells) than the αβ T cells.

The first signal in activation of T cells is provided by binding of theT cell receptor in a short peptide presented by the MHC on another cell.This ensures that only a T cell with a TCR specific to that peptide isactivated. The partner cell is usually an antigen presenting cell suchas a professional antigen presenting cell, usually a dendritic cell inthe case of naive responses, although B cells and macrophages can beimportant APCs.

According to the present invention, a molecule is capable of binding toa target if it has a significant affinity for said predetermined targetand binds to said predetermined target in standard assays. “Affinity” or“binding affinity” is often measured by equilibrium dissociationconstant (K_(D)). A molecule is not (substantially) capable of bindingto a target if it has no significant affinity for said target and doesnot bind significantly to said target in standard assays.

Cytotoxic T lymphocytes may be generated in vivo by incorporation of anantigen or a peptide fragment thereof into antigen-presenting cells invivo. The antigen or a peptide fragment thereof may be represented asprotein, as DNA (e.g. within a vector) or as RNA. The antigen may beprocessed to produce a peptide partner for the MHC molecule, while afragment thereof may be presented without the need for furtherprocessing. The latter is the case in particular, if these can bind toMHC molecules. In general, administration to a patient by intradermalinjection is possible. However, injection may also be carried outintranodally into a lymph node (Maloy et al. (2001), Proc Natl Acad SciUSA 98:3299-303). The resulting cells present the complex of interestand are recognized by autologous cytotoxic T lymphocytes which thenpropagate.

Specific activation of CD4+ or CD8+ T cells may be detected in a varietyof ways. Methods for detecting specific T cell activation includedetecting the proliferation of T cells, the production of cytokines(e.g., lymphokines), or the generation of cytolytic activity. For CD4+ Tcells, a preferred method for detecting specific T cell activation isthe detection of the proliferation of T cells. For CD8+ T cells, apreferred method for detecting specific T cell activation is thedetection of the generation of cytolytic activity.

By “cell characterized by presentation of an antigen” or “cellpresenting an antigen” or similar expressions is meant a cell such as adiseased cell, e.g. a cancer cell, or an antigen presenting cellpresenting the antigen it expresses or a fragment derived from saidantigen, e.g. by processing of the antigen, in the context of MHCmolecules, in particular MHC Class I molecules. Similarly, the terms“disease characterized by presentation of an antigen” denotes a diseaseinvolving cells characterized by presentation of an antigen, inparticular with class I MHC. Presentation of an antigen by a cell may beeffected by transfecting the cell with a nucleic acid such as RNAencoding the antigen.

By “fragment of an antigen which is presented” or similar expressions ismeant that the fragment can be presented by MHC class I or class II,preferably MHC class I, e.g. when added directly to antigen presentingcells. In one embodiment, the fragment is a fragment which is naturallypresented by cells expressing an antigen.

The term “immunologically equivalent” means that the immunologicallyequivalent molecule such as the immunologically equivalent amino acidsequence exhibits the same or essentially the same immunologicalproperties and/or exerts the same or essentially the same immunologicaleffects, e.g., with respect to the type of the immunological effect suchas induction of a humoral and/or cellular immune response, the strengthand/or duration of the induced immune reaction, or the specificity ofthe induced immune reaction. In the context of the present invention,the term “immunologically equivalent” is preferably used with respect tothe immunological effects or properties of a peptide used forimmunization. For example, an amino acid sequence is immunologicallyequivalent to a reference amino acid sequence if said amino acidsequence when exposed to the immune system of a subject induces animmune reaction having a specificity of reacting with the referenceamino acid sequence.

The term “immune effector functions” in the context of the presentinvention includes any functions mediated by components of the immunesystem that result, for example, in the killing of tumor cells, or inthe inhibition of tumor growth and/or inhibition of tumor development,including inhibition of tumor dissemination and metastasis. Preferably,the immune effector functions in the context of the present inventionare T cell mediated effector functions. Such functions comprise in thecase of a helper T cell (CD4+ T cell) the recognition of an antigen oran antigen fragment in the context of MHC class II molecules by T cellreceptors, the release of cytokines and/or the activation of CD8⁺lymphocytes (CTLs) and/or B-cells, and in the case of CTL therecognition of an antigen or an antigen fragment in the context of MHCclass I molecules by T cell receptors, the elimination of cellspresented in the context of MHC class I molecules, i.e., cellscharacterized by presentation of an antigen with class I MHC, forexample, via apoptosis or perform-mediated cell lysis, production ofcytokines such as IFN-γ and TNF-α, and specific cytolytic killing ofantigen expressing target cells.

According to the invention, the term “score” relates to a result,usually expressed numerically, of a test or examination. Terms such as“score better” or “score best” relate to a better result or the bestresult of a test or examination.

According to the invention, modified peptides are scored according totheir predicted ability to bind to MHC class II and according lo theexpression or abundance of the modified proteins from which the modifiedpeptides are derived. In general, a peptide with a predicted higherability to bind to MHC class II is scored belter than a peptide with apredicted lower ability to bind to MHC class II. Furthermore, a peptidewith higher expression or abundance of the corresponding modifiedprotein is scored better than a peptide with lower expression orabundance of the corresponding modified protein.

Terms such as “predict”, “predicting” or “prediction” relate to thedetermination of a likelihood.

According to the invention, ascertaining a score for binding of apeptide to one or more MHC class II molecules includes determining thelikelihood of binding of a peptide to one or more MHC class IImolecules.

A score for binding of a peptide to one or more MHC class II moleculesmay be ascertained by using any peptide: MHC binding predictive tools.For example, the immune epitope database analysis resource (IEDB-AR:http://tools.iedb.org) may be used.

Predictions are usually made against a set of MHC class II moleculessuch as a set of different MHC class II alleles such as all possible MHCclass II alleles or a set or subset of MHC class II alleles found in apatient. Preferably, the patient has the modification(s) theimmunogenicity of which is to be determined according to the inventionor which are to be selected and/or ranked according to their predictedimmunogenicity according to the invention. Preferably, the vaccinedescribed herein is to be provided ultimately for said patient.Accordingly, the present invention may also include determining the MHCclass II expression pattern of a patient.

The present invention also may comprise performing the method of theinvention on different peptides comprising the same modification(s)and/or different modifications.

The term “different peptides comprising the same modification(s)” in oneembodiment relates to peptides comprising or consisting of differentfragments of a modified protein, said different fragments comprising thesame modification(s) present in the protein but differing in lengthand/or position of the modification(s). If a protein has a modificationat position x, two or more fragments of said protein each comprising adifferent sequence window of said protein covering said position x areconsidered different peptides comprising the same modification(s).

The term “different peptides comprising different modifications” in oneembodiment relates to peptides either of the same and/or differinglengths comprising different modifications of either of the same and/ordifferent proteins. If a protein has modifications at positions x and y,two fragments of said protein each comprising a sequence window of saidprotein covering either position x or position y are considereddifferent peptides comprising different modifications.

The present invention also may comprise breaking of protein sequenceshaving modifications the immunogenicity of which is to be determinedaccording to the invention or which are to be selected and/or rankedaccording to their predicted immunogenicity according to the inventioninto appropriate peptide lengths for MHC binding and ascertaining scoresfor binding to one or more MHC class II molecules of different modifiedpeptides comprising the same and/or different modifications of eitherthe same and/or different proteins. Outputs may be ranked and mayconsist of a list of peptides and their predicted scores, indicatingtheir likelihood of binding.

The step of ascertaining a score for expression or abundance of themodified protein may be performed with all different modifications, asubset thereof, e.g. those modifications scoring best for binding to oneor more MHC class II molecules, or only with the modification scoringbest for binding to one or more MHC class II molecules.

Following said further step, the results may be ranked and may consistof a list of peptides and their predicted scores, indicating theirlikelihood of being immunogenic.

According to the invention, ascertaining a score for expression orabundance of the modified protein may be performed for a patient such asa cancer patient, for example, on a tumor specimen of a patient such asa cancer patient.

According to the invention, ascertaining a score for expression orabundance of a modified protein may comprises determining the level ofexpression of the protein to which the modification is associated and/ordetermining the level of expression of RNA encoding the protein to whichthe modification is associated (which again may be indicative for thelevel of expression of the protein to which the modification isassociated) and determining the frequency of the modified protein amongthe protein to which the modification is associated and/or determiningthe frequency of RNA encoding the modified protein among the RNAencoding the protein to which the modification is associated.

The frequency of the modified protein among the protein to which themodification is associated and/or the frequency of RNA encoding themodified protein among the RNA encoding the protein to which themodification is associated may be considered the proportion of themodified protein within the protein to which the modification isassociated and/or the proportion of RNA encoding the modified proteinwithin the RNA encoding the protein to which the modification isassociated.

According to the invention, the term “protein to which the modificationis associated” relates to the protein which may comprise themodification and includes the protein in its unmodified as well asmodified state.

According to the invention. the term “level of expression” may refer toan absolute or relative amount.

The amino acid modifications the immunogenicity of which is to bedetermined according to the present invention or which are to beselected and/or ranked according to their predicted immunogenicityaccording to the invention may result from mutations in the nucleic acidof a cell. Such mutations may be identified by known sequencingtechniques.

In one embodiment, the mutations arc cancer specific somatic mutationsin a tumor specimen of a cancer patient which may be determined byidentifying sequence differences between the genome, exome and/ortranscriptome of a tumor specimen and the genome, exome and/ortranscriptome of a non-tumorigenous specimen.

According to the invention a tumor specimen relates to any sample suchas a bodily sample derived from a patient containing or being expectedof containing tumor or cancer cells. The bodily sample may be any tissuesample such as blood, a tissue sample obtained from the primary tumor orfrom tumor metastases or any other sample containing tumor or cancercells. Preferably, a bodily sample is blood and cancer specific somaticmutations or sequence differences are determined in one or morecirculating tumor cells (CTCs) contained in the blood. In anotherembodiment, a tumor specimen relates to one or more isolated tumor orcancer cells such as circulating tumor cells (CTCs) or a samplecontaining one or more isolated tumor or cancer cells such ascirculating tumor cells (CTCs).

A non-tumorigenous specimen relates to any sample such as a bodilysample derived from a patient or another individual which preferably isof the same species as the patient, preferably a healthy individual notcontaining or not being expected of containing tumor or cancer cells.The bodily sample may be any tissue sample such as blood or a samplefrom a non-tumorigenous tissue.

The invention may involve the determination of the cancer mutationsignature of a patient. The term “cancer mutation signature” may referto all cancer mutations present in one or more cancer cells of a patientor it may refer to only a portion of the cancer mutations present in oneor more cancer cells of a patient. Accordingly, the present inventionmay involve the identification of all cancer specific mutations presentin one or more cancer cells of a patient or it may involve theidentification of only a portion of the cancer specific mutationspresent in one or more cancer cells of a patient. Generally, the methodsof the invention provides for the identification of a number ofmutations which provides a sufficient number of modifications ormodified peptides to be included in the methods of the invention.

Preferably, the mutations identified according to the present inventionare non-synonymous mutations, preferably non-synonymous mutations ofproteins expressed in a tumor or cancer cell.

In one embodiment, cancer specific somatic mutations or sequencedifferences are determined in the genome, preferably the entire genome,of a tumor specimen. Thus, the invention may comprise identifying thecancer mutation signature of the genome, preferably the entire genome ofone or more cancer cells. In one embodiment, the step of identifyingcancer specific somatic mutations in a tumor specimen of a cancerpatient comprises identifying the genome-wide cancer mutation profile.

In one embodiment, cancer specific somatic mutations or sequencedifferences are determined in the exome, preferably the entire exome, ofa tumor specimen. Thus, the invention may comprise identifying thecancer mutation signature of the exome, preferably the entire exome ofone or more cancer cells. In one embodiment, the step of identifyingcancer specific somatic mutations in a tumor specimen of a cancerpatient comprises identifying the exome-wide cancer mutation profile.

In one embodiment, cancer specific somatic mutations or sequencedifferences are determined in the transcriptome, preferably the entiretranscriptome, of a tumor specimen. Thus, the invention may compriseidentifying the cancer mutation signature of the transcriptome,preferably the entire transcriptome of one or more cancer cells. In oneembodiment, the step of identifying cancer specific somatic mutations ina tumor specimen of a cancer patient comprises identifying thetranscriptome-wide cancer mutation profile.

In one embodiment, the step of identifying cancer specific somaticmutations or identifying sequence differences comprises single cellsequencing of one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or even more cancer cells. Thus, theinvention may comprise identifying a cancer mutation signature of saidone or more cancer cells. In one embodiment, the cancer cells arecirculating tumor cells. The cancer cells such as the circulating tumorcells may be isolated prior to single cell sequencing.

In one embodiment, the step of identifying cancer specific somaticmutations or identifying sequence differences involves using nextgeneration sequencing (NGS).

In one embodiment, the step of identifying cancer specific somaticmutations or identifying sequence differences comprises sequencinggenomic DNA and/or RNA of the tumor specimen.

To reveal cancer specific somatic mutations or sequence differences thesequence information obtained from the tumor specimen is preferablycompared with a reference such as sequence information obtained fromsequencing nucleic acid such as DNA or RNA of normal non-cancerous cellssuch as germline cells which may either be obtained from the patient ora different individual. In one embodiment, normal genomic germline DNAis obtained from peripheral blood mononuclear cells (PBMCs)

The term “genome” relates to the total amount of genetic information inthe chromosomes of an organism or a cell.

The term “exome” refers to part of the genome of an organism formed byexons, which are coding portions of expressed genes. The exome providesthe genetic blueprint used in the synthesis of proteins and otherfunctional gene products. It is the most functionally relevant part ofthe genome and, therefore, it is most likely to contribute to thephenotype of an organism. The exome of the human genome is estimated tocomprise 1.5% of the total genome (Ng, P C et al., PLoS Gen., 4(8):1-15, 2008).

The term “transcriptome” relates to the set of all RNA molecules,including mRNA, rRNA, tRNA, and other non-coding RNA produced in onecell or a population of cells. In context of the present invention thetranscriptome means the set of all RNA molecules produced in one cell, apopulation of cells, preferably a population of cancer cells, or allcells of a given individual at a certain time point.

A “nucleic acid” is according to the invention preferablydeoxyribonucleic acid (DNA) or ribonucleic acid (RNA). more preferablyRNA, most preferably in vitro transcribed RNA (IVT RNA) or syntheticRNA. Nucleic acids include according to the invention genomic DNA, cDNA,mRNA, recombinantly produced and chemically synthesized molecules.According to the invention, a nucleic acid may be present as asingle-stranded or double-stranded and linear or covalently circularlyclosed molecule. A nucleic acid can, according to the invention, beisolated. The term “isolated nucleic acid” means, according to theinvention, that the nucleic acid (i) was amplified in vitro, for examplevia polymerase chain reaction (PCR), (ii) was produced recombinantly bycloning, (iii) was purified, for example, by cleavage and separation bygel electrophoresis, or (iv) was synthesized, for example, by chemicalsynthesis. A nucleic can be employed for introduction into, i.e.transfection of, cells, in particular, in the form of RNA which can beprepared by in vitro transcription from a DNA template. The RNA canmoreover be modified before application by stabilizing sequences,capping, and polyadenylation.

The term “genetic material” refers to isolated nucleic acid, either DNAor RNA, a section of a double helix, a section of a chromosome, or anorganism's or cell's entire genome, in particular its exome ortranscriptome.

The term “mutation” refers to a change of or difference in the nucleicacid sequence (nucleotide substitution, addition or deletion) comparedto a reference. A “somatic mutation” can occur in any of the cells ofthe body except the germ cells (sperm and egg) and therefore are notpassed on to children. These alterations can (but do not always) causecancer or other diseases.

Preferably a mutation is a non-synonymous mutation. The term“non-synonymous mutation” refers to a mutation, preferably a nucleotidesubstitution, which does result in an amino acid change such as an aminoacid substitution in the translation product.

According to the invention, the term “mutation” includes pointmutations, Indels, fusions, chromothripsis and RNA edits.

According to the invention, the term “Indel” describes a specialmutation class, defined as a mutation resulting in a colocalizedinsertion and deletion and a net gain or loss in nucleotides. In codingregions of the genome, unless the length of an indel is a multiple of 3,they produce a frameshift mutation. Indels can be contrasted with apoint mutation; where an Indel inserts and deletes nucleotides from asequence, a point mutation is a form of substitution that replaces oneof the nucleotides.

Fusions can generate hybrid genes formed from two previously separategenes. It can occur as the result of a translocation, interstitialdeletion, or chromosomal inversion. Often, fusion genes are oncogenes.Oncogenic fusion genes may lead to a gene product with a new ordifferent function from the two fusion partners. Alternatively, aproto-oncogene is fused to a strong promoter, and thereby the oncogenicfunction is set to function by an upregulation caused by the strongpromoter of the upstream fusion partner. Oncogenic fusion transcriptsmay also be caused by trans-splicing or read-through events.

According to the invention, the term “chromothripsis” refers to agenetic phenomenon by which specific regions of the genome are shatteredand then stitched together via a single devastating event.

According to the invention, the term “RNA edit” or “RNA editing” refersto molecular processes in which the information content in an RNAmolecule is altered through a chemical change in the base makeup. RNAediting includes nucleoside modifications such as cytidine (C) touridine (U) and adenosine (A) to inosine (I) deaminations, as well asnon-templated nucleotide additions and insertions. RNA editing in mRNAseffectively alters the amino acid sequence of the encoded protein sothat it differs from that predicted by the genomic DNA sequence.

The term “cancer mutation signature” refers to a set of mutations whichare present in cancer cells when compared to non-cancerous referencecells.

According to the invention, a “reference” may be used to correlate andcompare the results obtained in the methods of the invention from atumor specimen. Typically the “reference” may be obtained on the basisof one or more normal specimens, in particular specimens which are notaffected by a cancer disease, either obtained from a patient or one ormore different individuals, preferably healthy individuals, inparticular individuals of the same species. A “reference” can bedetermined empirically by testing a sufficiently large number of normalspecimens.

Any suitable sequencing method can be used according to the inventionfor determining mutations, Next Generation Sequencing (NGS) technologiesbeing preferred. Third Generation Sequencing methods might substitutefor the NGS technology in the future to speed up the sequencing step ofthe method. For clarification purposes: the terms “Next GenerationSequencing” or “NGS” in the context of the present invention mean allnovel high throughput sequencing technologies which, in contrast to the“conventional” sequencing methodology known as Sanger chemistry, readnucleic acid templates randomly in parallel along the entire genome bybreaking the entire genome into small pieces. Such NGS technologies(also known as massively parallel sequencing technologies) are able todeliver nucleic acid sequence information of a whole genome, exome,transcriptome (all transcribed sequences of a genome) or methylome (allmethylated sequences of a genome) in very short time periods, e.g.within 1-2 weeks, preferably within 1-7 days or most preferably withinless than 24 hours and allow, in principle, single cell sequencingapproaches. Multiple NGS platforms which arc commercially available orwhich are mentioned in the literature can be used in the context of thepresent invention e.g. those described in detail in Zhang el at. 2011:The impact of next-generation sequencing on genomics. J. Genet Genomics38 (3), 95-109; or in Voelkerding et al. 2009: Next generationsequencing: From basic research to diagnostics. Clinical chemistry 55,641-658. Non-limiting examples of such NGS technologies/platforms are

-   -   1) The sequencing-by-synthesis technology known as        pyrosequencing implemented e.g. in the GS-FLX 454 Genome        Sequencer™ of Roche-associated company 454 Life Sciences        (Branford, Conn.), first described in Ronaghi et al. 1998: A        sequencing method bused on real-time pyrophosphate”. Science 281        (5375), 363-365. This technology uses an emulsion PCR in which        single-stranded DNA binding beads are encapsulated by vigorous        vortexing into aqueous micelles containing PCR reactants        surrounded by oil for emulsion PCR amplification. During the        pyrosequencing process, light emitted from phosphate molecules        during nucleotide incorporation is recorded as the polymerase        synthesizes the DNA strand.    -   2) The sequencing-by-synthesis approaches developed by Solexa        (now part of Illumina Inc., San Diego, Calif.) which is based on        reversible dye-terminators and implemented e.g. in the        Illumina/Solexa Genome Analyzer™ and in the Illumina HiSeq 2000        Genome Analyzer™. In this technology, all four nucleotides are        added simultaneously into oligo-primed cluster fragments in        flow-cell channels along with DNA polymerase. Bridge        amplification extends cluster strands with all four        fluorescently labeled nucleotides for sequencing.    -   3) Sequencing-by-ligation approaches, e.g. implemented in the        SOLid™ platform of Applied Biosystems (now Life Technologies        Corporation, Carlsbad, Calif.). In this technology, a pool of        all possible oligonucleotides of a fixed length are labeled        according to the sequenced position. Oligonucleotides are        annealed and ligated; the preferential ligation by DNA ligase        for matching sequences results in a signal informative of the        nucleotide at that position. Before sequencing, the DNA is        amplified by emulsion PCR. The resulting bead, each containing        only copies of the same DNA molecule, are deposited on a glass        slide. As a second example, he Polonator™ G.007 platform of        Dover Systems (Salem, N.H.) also employs a        sequencing-by-ligation approach by using a randomly arrayed,        bead-based, emulsion PCR to amplify DNA fragments for parallel        sequencing.    -   4) Single-molecule sequencing technologies such as e.g.        implemented in the PacBio RS system of Pacific Biosciences        (Menlo Park, Calif.) or in the HeliScope™ platform of Helicos        Biosciences (Cambridge, Mass.). The distinct characteristic of        this technology is its ability to sequence single DNA or RNA        molecules without amplification, defined as Single-Molecule Real        Time (SMRT) DNA sequencing. For example, HeliScope uses a highly        sensitive fluorescence detection system to directly detect each        nucleotide as it is synthesized. A similar approach based on        fluorescence resonance energy transfer (FRET) has been developed        from Visigen Biotechnology (Houston, Tex.). Other        fluorescence-based single-molecule techniques are from U.S.        Genomics (GeneEngine™) and Genovoxx (AnyGene™).    -   5) Nano-technologies for single-molecule sequencing in which        various nanostructures are used which are e.g. arranged on a        chip to monitor the movement of a polymerase molecule on a        single strand during replication. Non-limiting examples for        approaches based on nano-technologies arc the GridON™ platform        of Oxford Nanopore Technologies (Oxford, UK), the        hybridization-assisted nano-pore sequencing (HANS™) platforms        developed by Nabsys (Providence, R.I.), and the proprietary        ligase-based DNA sequencing platform with DNA nanoball (DNB)        technology called combinatorial probe-anchor ligation (cPAL™).    -   6) Electron microscopy based technologies for single-molecule        sequencing, e.g. those developed by LightSpeed Genomics        (Sunnyvale, Calif.) and Haleyon Molecular (Redwood City, Calif.)    -   7) Ion semiconductor sequencing which is based on the detection        of hydrogen ions that are released during the polymerisation of        DNA. For example, Ion Torrent Systems (San Francisco, Calif.)        uses a high-density array of micro-machined wells to perform        this biochemical process in a massively parallel way. Each well        holds a different UNA template. Beneath the wells is an        ion-sensitive layer and beneath that a proprietary Ion sensor.

Preferably, DNA and RNA preparations serve as starting material for NGS.Such nucleic acids can be easily obtained from samples such asbiological material, e.g. from fresh, flash-frozen or formalin-fixedparaffin embedded tumor tissues (FFPE) or from freshly isolated cells orfrom CTCs which are present in the peripheral blood of patients. Normalnon-mutated genomic DNA or RNA can be extracted from normal, somatictissue, however germline cells are preferred in the context of thepresent invention. Germline DNA or RNA may be extracted from peripheralblood mononuclear cells (PBMCs) in patients with non-hematologicalmalignancies. Although nucleic acids extruded from FFPE tissues orfreshly isolated single cells are highly fragmented, they are suitablefor NGS applications.

Several targeted NGS methods for exome sequencing are described in theliterature (for review see e.g. Teer and Mullikin 2010: Human Mol Genet19 (21 R145-51), all of which can be used in conjunction with thepresent invention. Many of these methods (described e.g. as genomecapture, genome partitioning, genome enrichment etc.) use hybridizationtechniques and include array-based (e.g. Hodges et al. 2007: Nat. Genet.39, 1522-1527) and liquid-based (e.g. Choi el al. 2009: Proc. Natl.Acad. Sci USA 106, 19096-19101) hybridization approaches. Commercialkits for DNA sample preparation and subsequent exome capture are alsoavailable: for example, Illumina Inc. (San Diego, Calif.) offers theTruSeq™ DNA Sample Preparation Kit and the Exome Enrichment Kit TruSeq™Exome Enrichment Kit.

In order to reduce the number of false positive findings in detectingcancer specific somatic mutations or sequence differences when comparinge.g. the sequence of a tumor sample to the sequence of a referencesample such as the sequence of a germ line sample it is preferred todetermine the sequence in replicates of one or both of these sampletypes. Thus, it is preferred that the sequence of a reference samplesuch as the sequence of a germ line sample is determined twice, threetimes or more. Alternatively or additionally, the sequence of a tumorsample is determined twice, three times or more. It may also be possibleto determine the sequence of a reference sample such as the sequence ofa germ line sample and/or the sequence of a tumor sample more than onceby determining at least once the sequence in genomic DNA and determiningat least once the sequence in RNA of said reference sample and/or ofsaid tumor sample. For example, by determining the variations betweenreplicates of a reference sample such as a germ line sample the expectedrate of false positive (FDR) somatic mutations as a statistical quantitycan be estimated. Technical repeats of a sample should generateidentical results and any detected mutation in this “same vs. samecomparison” is a false positive. In particular, to determine the falsediscovery rate for somatic mutation detection in a tumor sample relativeto a reference sample, a technical repeat of the reference sample can beused as a reference to estimate the number of false positives.Furthermore, various quality related metrics (e.g. coverage or SNPquality) may be combined into a single quality score using a machinelearning approach. For a given somatic variation all other variationswith an exceeding quality score may be counted, which enables a rankingof all variations in a dataset.

In the context of the present invention, the term “RNA” relates to amolecule which comprises at least one ribonucleotide residue andpreferably being entirely or substantially composed of ribonucleotideresidues. “Ribonucleotide” relates to a nucleotide with a hydroxyl groupat the 2′-position of a β-D-ribofuranosyl group. The term “RNA”comprises double-stranded RNA, single-stranded RNA, isolated RNA such aspartially or completely purified RNA, essentially pure RNA, syntheticRNA, and recombinantly generated RNA such as modified RNA which differsfrom naturally occurring RNA by addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of a RNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in RNA molecules can also comprise non-standard nucleotides,such as non-naturally occurring nucleotides or chemically synthesizednucleotides or deoxynucleotides. These altered RNAs can be referred toas analogs or analogs of naturally-occurring RNA.

According to the present invention, the term “RNA” includes andpreferably relates to “mRNA”. The term “mRNA” means “messenger-RNA” andrelates to a “transcript” which is generated by using a DNA template andencodes a peptide or polypeptide. Typically, an mRNA comprises a 5′-UTR,a protein coding region, and a 3′-UTR, mRNA only possesses limitedhalf-life in cells and in vitro. In the context of the presentinvention, mRNA may be generated by in vitro transcription from a DNAtemplate. The in vitro transcription methodology is known to the skilledperson. For example, there is a variety of in vitro transcription kitscommercially available.

According to the invention, the stability and translation efficiency ofRNA may be modified as required. For example. RNA may be stabilized andits translation increased by one or more modifications having astabilizing effects and/or increasing translation efficiency of RNA.Such modifications arc described, for example, in PCT/EP2006/009448incorporated herein by reference. In order to increase expression of theRNA used according to the present invention, it may be modified withinthe coding region, i.e. the sequence encoding the expressed peptide orprotein, preferably without altering the sequence of the expressedpeptide or protein, so as to increase the GC-content to increase mRNAstability and to perform a codon optimization and, thus, enhancetranslation in cells.

The term “modification” in the context of the RNA used in the presentinvention includes any modification of an RNA which is not naturallypresent in said RNA.

In one embodiment of the invention, the RNA used according to theinvention does not have uncapped 5′-triposphates. Removal of suchuncapped 5′-triphosphates can be achieved by treating RNA with aphosphatase.

The RNA according to the invention may have modified ribonucleotides inorder to increase its stability and/or decrease cytotoxicity. Forexample, in one embodiment, in the RNA used according to the invention5-methylcylidine is substituted partially or completely, preferablycompletely, for cytidine. Alternatively or additionally, in oneembodiment, in the RNA used according to the invention pseudouridine issubstituted partially or completely, preferably completely, for uridine.

In one embodiment, the term “modification” relates to providing an RNAwith a 5′-cap or 5′-cap analog. The term “5′-cap” refers to a capstructure found on the 5′-end of an mRNA molecule and generally consistsof a guanosine nucleotide connected to the mRNA via an unusual 5′ to 5′triphosphate linkage. In one embodiment, this guanosine is methylated atthe 7-position. The term “conventional 5′-cap” refers to a naturallyoccurring RNA 5′-cap, preferably to the 7-methylguanosine cap (m⁷G). Inthe context of the present invention, the term “5′-cap” includes a5′-cap analog that resembles the RNA cap structure and is modified topossess the ability to stabilize RNA and/or enhance translation of RNAif attached thereto, preferably in vivo and/or in a cell.

Providing an RNA with a 5′-cap or 5′-cap analog may be achieved by invitro transcription of a DNA template in presence of said 5′-cap or5′-cap analog, wherein said 5-cap is co-transcriptionally incorporatedinto the generated RNA strand, or the RNA may be generated, for example,by in vitro transcription, and the 5′-cap may be attached to the RNApost-transcriptionally using capping enzymes, for example, cappingenzymes of vaccinia virus.

The RNA may comprise further modifications. For example, a furthermodification of the RNA used in the present invention may be anextension or truncation of the naturally occurring poly(A) tail or analteration of the 5′- or 3′-untranslated regions (UTR) such asintroduction of a UTR which is not related to the coding region of saidRNA, for example, the exchange of the existing 3′-UTR with or theinsertion of one or more, preferably two copies of a 3′-UTR derived froma globin gene, such as alpha2-globin, alpha 1-globin, beta-globin,preferably beta-globin, more preferably human beta-globin.

RNA having an unmasked poly-A sequence is translated more efficientlythan RNA having a masked poly-A sequence. The term “poly(A) tail” or“poly-A sequence” relates to a sequence of adenyl (A) residues whichtypically is located on the 3′-end of a RNA molecule and “unmaskedpoly-A sequence” means that the poly-A sequence at the 3′ end of an RNAmolecule ends with an A of the poly-A sequence and is not followed bynucleotides other than A located at the 3′ end i.e. downstream, of thepoly-A sequence. Furthermore, a long poly-A sequence of about 120 basepairs results in an optimal transcript stability and translationefficiency of RNA.

Therefore, in order to increase stability and/or expression of the RNAused according to the present invention, it may be modified so as to bepresent in conjunction with a poly-A sequence, preferably having alength of 10 to 500, more preferably 30 to 300, even more preferably 65to 200 and especially 100 to 150 adenosine residues. In an especiallypreferred embodiment the poly-A sequence has a length of approximately120 adenosine residues. To further increase stability and/or expressionof the RNA used according to the invention, the poly-A sequence can beunmasked.

In addition, incorporation of a 3′-non translated region (UTR) into the3′-non translated region of an RNA molecule can result in an enhancementin translation efficiency. A synergistic effect may be achieved byincorporating two or more of such 3′-non translated regions. The 3′-nontranslated regions may be autologous or heterologous to the RNA intowhich they are introduced.

In line particular embodiment the 3′-non translated region is derivedfrom the human β-globin gene.

A combination of the above described modifications, i.e. incorporationof a poly-A sequence, unmasking of a poly-A sequence and incorporationof one or more 3′-non translated regions, has a synergistic influence onthe stability of RNA and increase in translation efficiency.

The term “stability” of RNA relates to the “half-life” of RNA.“Half-life” relates to the period of time which is needed to eliminatehalf of the activity, amount, or number of molecules. In the context ofthe present invention, the half-life of an RNA is indicative for thestability of said RNA. The half-life of RNA may influence the “durationof expression” of the RNA. It can be expected that RNA having a longhalf-life will be expressed for an extended time period.

Of course, if according to the present invention it is desired todecrease stability and/or translation efficiency of RNA, it is possibleto modify RNA so as to interfere with the function of elements asdescribed above increasing the stability and/or translation efficiencyof RNA.

The term “expression” is used according to the invention in its mostgeneral meaning and comprises the production of RNA and/or peptides,polypeptides or proteins, e.g. by transcription and/or translation. Withrespect to RNA, the term “expression” or “translation” relates inparticular to the production of peptides, polypeptides or proteins. Italso comprises partial expression of nucleic acids. Moreover, expressioncan be transient or stable.

According to the invention, the term expression also includes an“aberrant expression” or “abnormal expression”. “Aberrant expression” or“abnormal expression” means according to the invention that expressionis altered, preferably increased, compared to a reference, e.g. a statein a subject not having a disease associated with aberrant or abnormalexpression of a certain protein, e.g., a tumor antigen. An increase inexpression refers to an increase by at least 10%, in particular at least20%, at least 50% or at least 100%, or more. In one embodiment,expression is only found in a diseased tissue, while expression in ahealthy tissue is repressed.

The term “specifically expressed” means that a protein is essentiallyonly expressed in a specific tissue or organ. For example, a tumorantigen specifically expressed in gastric mucosa means that said proteinis primarily expressed in gastric mucosa and is not expressed in othertissues or is not expressed to a significant extent in other tissue ororgan types. Thus, a protein that is exclusively expressed in cells ofthe gastric mucosa and to a significantly lesser extent in any othertissue, such us testis, is specifically expressed in cells of thegastric mucosa. In some embodiments, a tumor antigen may also bespecifically expressed under normal conditions in more than one tissuetype or organ, such as in 2 or 3 tissue types or organs, but preferablyin not more than 3 different tissue or organ types. In this case, thetumor antigen is then specifically expressed in these organs. Forexample, if a tumor antigen is expressed under normal conditionspreferably lo an approximately equal extent in lung and stomach, saidtumor antigen is specifically expressed in lung and stomach.

In the context of the present invention, the term “transcription”relates to a process, wherein the genetic code in a DNA sequence istranscribed into RNA. Subsequently, the RNA may be translated intoprotein. According to the present invention, the term “transcription”comprises “in vitro transcription”, wherein the term “in vitrotranscription” relates to a process wherein RNA. in particular mRNA, isin vitro synthesized in a cell-free system, preferably using appropriatecell extracts. Preferably, cloning vectors are applied for thegeneration of transcripts. These cloning vectors are generallydesignated as transcription vectors and are according to the presentinvention encompassed by the term “vector”. According to the presentinvention, the RNA used in the present invention preferably is in vitrotranscribed RNA (IVT-RNA) and may be obtained by in vitro transcriptionof an appropriate DNA template. The promoter for controllingtranscription can be any promoter for any RNA polymerase. Particularexamples of RNA polymerases are the T7, T3, and SP6 RNA polymerases.Preferably, the in vitro transcription according to the invention iscontrolled by a T7 or SP6 promoter. A DNA template for in vitrotranscription may be obtained by cloning of a nucleic acid, inparticular cDNA, and introducing it into an appropriate vector for invitro transcription. The cDNA may be obtained by reverse transcriptionof RNA.

The term “translation” according lo the invention relates to the processin the ribosomes of a cell by which a strand of messenger RNA directsthe assembly of a sequence of amino acids to make a peptide, polypeptideor protein.

Expression control sequences or regulatory sequences, which according tothe invention may be linked functionally with a nucleic acid, can behomologous or heterologous with respect to the nucleic acid. A codingsequence and a regulatory sequence are linked together “functionally” ifthey are bound together covalently, so that the transcription ortranslation of the coding sequence is under the control or under theinfluence of the regulatory sequence. If the coding sequence is to betranslated into a functional protein, with functional linkage of aregulatory sequence with the coding sequence, induction of theregulatory sequence leads to a transcription of the coding sequence,without causing a reading frame shift in the coding sequence orinability of the coding sequence to be translated into the desiredprotein or peptide.

The term “expression control sequence” or “regulatory sequence”comprises, according to the invention, promoters, ribosome-bindingsequences and other control elements, which control the transcription ofa nucleic acid or the translation of the derived RNA. In certainembodiments of the invention, the regulatory sequences can becontrolled. The precise structure of regulatory sequences can varydepending on the species or depending on the cell type, but generallycomprises 5′-untranscribed and 5′- and 3′-untranslated sequences, whichare involved in the initiation of transcription or translation, such asTATA-box, capping-sequence, CAAT-sequence and the like. In particular,5′-untranscribed regulatory sequences comprise a promoter region thatincludes a promoter sequence for transcriptional control of thefunctionally bound gene. Regulatory sequences can also comprise enhancersequences or upstream activator sequences.

Preferably, according to the invention, RNA to be expressed in a cell isintroduced into said cell. In one embodiment of the methods according tothe invention, the RNA that is to be introduced into a cell is obtainedby in vitro transcription of an appropriate DNA template.

According to the invention, terms such as “RNA capable of expressing”and “RNA encoding” arc used interchangeably herein and with respect to aparticular peptide or polypeptide mean that the RNA, if present in theappropriate environment, preferably within a cell, can be expressed toproduce said peptide or polypeptide. Preferably, RNA according to theinvention is able to interact with the cellular translation machinery toprovide the peptide or polypeptide it is capable of expressing.

Terms such as “transferring”, “introducing” or “transfecting” are usedinterchangeably herein and relate to the introduction of nucleic acids,in particular exogenous or heterologous nucleic acids, in particular RNAinto a cell. According to the present invention, the cell can form partof an organ, a tissue and/or an organism. According to the presentinvention, the administration of a nucleic acid is either achieved asnaked nucleic acid or in combination with an administration reagent.Preferably, administration of nucleic acids is in the form of nakednucleic acids. Preferably, the RNA is administered in combination withstabilizing substances such as RNase inhibitors. The present inventionalso envisions the repeated introduction of nucleic acids into cells toallow sustained expression for extended time periods.

Cells can be transfected with any carriers with which RNA can beassociated, e.g. by forming complexes with the RNA or forming vesiclesin which the RNA is enclosed or encapsulated, resulting in increasedstability of the RNA compared to naked RNA. Carriers useful according tothe invention include, for example, lipid-containing carriers such asanionic lipids, liposomes, in particular cationic liposomes, andmicelles, and nanoparticles. Cationic lipids may form complexes withnegatively charged nucleic acids. Any cationic lipid may be usedaccording to the invention.

Preferably, the introduction of RNA which encodes a peptide orpolypeptide into a cell, in particular into a cell present in vivo,results in expression of said peptide or polypeptide in the cell. Inparticular embodiments, the targeting of the nucleic acids to particularcells is preferred. In such embodiments, a carrier which is applied forthe administration of the nucleic acid to a cell (for example, aretrovirus or a liposome), exhibits a targeting molecule. For example, amolecule such as an antibody which is specific for a surface membraneprotein on the target cell or a ligand for a receptor on the target cellmay be incorporated into the nucleic acid carrier or may be boundthereto. In case the nucleic acid is administered by liposomes, proteinswhich bind to a surface membrane protein which is associated withendocytosis may be incorporated into the liposome formulation in orderlo enable targeting and/or uptake. Such proteins encompass capsidproteins of fragments thereof which are specific for a particular celltype, antibodies against proteins which are internalized, proteins whichtarget an intracellular location etc.

The term “cell” or “host cell” preferably is an intact cell, i.e. a cellwith an intact membrane that has not released its normal intracellularcomponents such as enzymes, organelles, or genetic material. An intactcell preferably is a viable cell, i.e. a living cell capable of carryingout its normal metabolic functions. Preferably said term relatesaccording to the invention to any cell which can be transformed ortransfected with an exogenous nucleic acid. The term “cell” includesaccording to the invention prokaryotic cells (e.g., E. coli) oreukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells,K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells).The exogenous nucleic acid may be found inside the cell (i) freelydispersed as such, (ii) incorporated in a recombinant vector, or (iii)integrated into the host cell genome or mitochondrial DNA. Mammaliancells are particularly preferred, such as cells from humans, mice,hamsters, pigs, goats, and primates. The cells may be derived from alarge number of tissue types and include primary cells and cell lines.Specific examples include keratinocytes, peripheral blood leukocytes,bone marrow stem cells, and embryonic stem cells. In furtherembodiments, the cell is an antigen-presenting cell, in particular adendritic cell, a monocyte, or macrophage.

A cell which comprises a nucleic acid molecule preferably expresses thepeptide or polypeptide encoded by the nucleic acid.

The term “clonal expansion” refers lo a process wherein a specificentity is multiplied. In the context of the present invention, the termis preferably used in the context of an immunological response in whichlymphocytes are stimulated by an antigen, proliferate, and the specificlymphocyte recognizing said antigen is amplified. Preferably, clonalexpansion leads to differentiation of the lymphocytes.

Terms such as “reducing” or “inhibiting” relate to the ability to causean overall decrease, preferably of 5% or greater, 10% or greater, 20% orgreater, more preferably of 50% or greater, and most preferably of 75%or greater, in the level. The term “inhibit” or similar phrases includesa complete or essentially complete inhibition, i.e. a reduction to zeroor essentially to zero.

Terms such as “increasing”, “enhancing”, “promoting” or “prolonging”preferably relate lo an increase, enhancement, promotion or prolongationby about at least 10%, preferably at least 20%, preferably al least 30%,preferably at least 40%, preferably at least 50%, preferably at least80%, preferably at least 100%, preferably at least 200% and inparticular at least 300%. These terms may also relate to an increase,enhancement, promotion or prolongation from zero or a non-measurable ornon-delectable level to a level of more than zero or a level which ismeasurable or detectable.

The present invention provides vaccines such as cancer vaccines designedon the basis of amino acid modifications or modified peptides predictedas being immunogenic by the methods of the present invention.

According to the invention, the term “vaccine” relates to apharmaceutical preparation (pharmaceutical composition) or product thatupon administration induces an immune response, in particular a cellularimmune response, which recognizes and attacks a pathogen or a diseasedcell such as a cancer cell. A vaccine may be used for the prevention ortreatment of a disease. The term “personalized cancer vaccine” or“individualized cancer vaccine” concerns a particular cancer patient andmeans that a cancer vaccine is adapted lo the needs or specialcircumstances of an individual cancer patient.

In one embodiment, a vaccine provided according to the invention maycomprise a peptide or polypeptide comprising one or more amino acidmodifications or one or more modified peptides predicted as beingimmunogenic by the methods of the invention or a nucleic acid,preferably RNA, encoding said peptide or polypeptide.

The cancer vaccines provided according to the invention whenadministered to a patent provide one or more T cell epitopes suitablefor stimulating, priming and/or expanding T cells specific for thepatient's tumor. The T cells are preferably directed against cellsexpressing antigens from which the T cell epitopes are derived. Thus,the vaccines described herein are preferably capable of inducing orpromoting a cellular response, preferably cytotoxic T cell activity,against a cancer disease characterized by presentation of one or moretumor-associated neoantigens with class I MHC. Since a vaccine providedaccording to the present invention will target cancer specific mutationsit will be specific for the patient's tumor.

A vaccine provided according to the invention relates to a vaccine whichwhen administered to a patent preferably provides one or more T cellepitopes, such as 2 or more, 5 or more, 10 or more, 15 or more, 20 ormore, 25 or more, 30 or more and preferably up to 60, up to 55, up to50, up to 45, up to 40, up to 35 or up to 30 T cell epitopes,incorporating amino acid modifications or modified peptides predicted asbeing immunogenic by the methods of the invention. Such T cell epitopesare also termed “neo-epitopes” herein. Presentation of these epitopes bycells of a patient, in particular antigen presenting cells, preferablyresults in T cells targeting the epitopes when bound to MHC and thus,the patient's tumor, preferably the primary tumor as well as tumormetastases, expressing antigens from which the T cell epitopes arederived and presenting the same epitopes on the surface of the tumorcells.

The methods of the invention may comprise the further step ofdetermining the usability of the identified amino acid modifications ormodified peptides for cancer vaccination. Thus further steps can involveone or more of the following: (i) assessing whether the modificationsare located in known or predicted MHC presented epitopes, (ii) in vitroand/or in silico testing whether the modifications are located in MHCpresented epitopes, e.g. testing whether the modifications are part ofpeptide sequences which are processed into and/or presented as MHCpresented epitopes, and (iii) in vitro testing whether the envisagedmodified epitopes, in particular when present in their natural sequencecontext, e.g. when flanked by amino acid sequences also flanking saidepitopes in the naturally occurring protein, and when expressed inantigen presenting cells are able to stimulate T cells such as T cellsof the patient having the desired specificity. Such flanking sequenceseach may comprise 3 or more, 5 or more, 10 or more, 15 or more, 20 ormore and preferably up to 50, up to 45, up to 40, up to 35 or up to 30amino acids and may flank the epitope sequence N-terminally and/orC-terminally.

Modified peptides determined according to the invention may be rankedfor their usability as epitopes for cancer vaccination. Thus, in oneaspect, the method of the invention comprises a manual or computer-basedanalytical process in which the identified modified peptides areanalyzed and selected for their usability in the respective vaccine tobe provided. In a preferred embodiment, said analytical process is acomputational algorithm-based process. Preferably, said analyticalprocess comprises determining and/or ranking epitopes according to aprediction of their capacity of being immunogenic.

The neo-epitopes identified according to the invention and provided by avaccine of the invention are preferably present in the form of apolypeptide comprising said neo-epitopes such as a polyepitopicpolypeptide or a nucleic acid, in particular RNA, encoding saidpolypeptide. Furthermore, the neo-epitopes may be present in thepolypeptide in the form of a vaccine sequence, i.e. present in theirnatural sequence context, e.g. flanked by amino acid sequences alsoflanking said epitopes in the naturally occurring protein. Such flankingsequences each may comprise 5 or more, 10 or more, 15 or more, 20 ormore and preferably up to 50, up to 45, up to 40, up to 35 or up to 30amino acids and may flank the epitope sequence N-terminally and/orC-terminally. Thus, a vaccine sequence may comprise 20 or more, 25 ormore, 30 or more, 35 or more, 40 or more and preferably up to 50, up to45, up to 40, up to 35 or up to 30 amino acids. In one embodiment, theneo-epitopes and/or vaccine sequences are lined up in the polypeptidehead-to-tail.

In one embodiment, the neo-epitopes and/or vaccine sequences are spacedby linkers, in particular neutral linkers. The term “linker” accordingto the invention relates to a peptide added between two peptide domainssuch as epitopes or vaccine sequences to connect said peptide domains.There is no particular limitation regarding the linker sequence.However, it is preferred that the linker sequence reduces sterichindrance between the two peptide domains, is well translated, andsupports or allows processing of the epitopes. Furthermore, the linkershould have no or only little immunogenic sequence elements. Linkerspreferably should not create non-endogenous neo-epitopes like thosegenerated from the junction suture between adjacent neo-epitopes, whichmight generate unwanted immune reactions. Therefore, the polyepitopicvaccine should preferably contain linker sequences which are able toreduce the number of unwanted MHC binding junction epitopes. Hoyt et al.(EMBO J. 25(8), 1720-9, 2006) and Zhang el al. (J. Biol. Chem., 279(10),8635-41, 2004) have shown that glycine-rich sequences impair proteasomalprocessing and thus the use of glycine rich linker sequences act tominimize the number of linker-contained peptides that can be processedby the proteasome. Furthermore, glycine was observed to inhibit a strongbinding in MHC binding groove positions (Abastado et al., J. Immunol.151(7), 3569-75, 1993). Schlessinger et al. (Proteins, 61(1), 115-26,2005) had found that amino acids glycine and serine included in an aminoacid sequence result in a more flexible protein that is more efficientlytranslated and processed by the proteasome, enabling better access tothe encoded neo-epitopes. The linker each may comprise 3 or more, 6 ormore, 9 or more, 10 or more, 15 or more, 20 or more and preferably up to50, up to 45, up to 40, up to 35 or up to 30 amino acids. Preferably thelinker is enriched in glycine and/or serine amino acids. Preferably, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% of the amino acids of the linker are glycine and/or serine. Inone preferred embodiment, a linker is substantially composed of theamino acids glycine and serine. In one embodiment, the linker comprisesthe amino acid sequence (GGS)_(a)(GSS)_(b)(GGG)_(c)(SSG)_(d)(GSG)_(e)wherein a, b, c, d and e is independently a number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20(SEQ ID NO: 91) and wherein a+b+c+d+e are different from 0 andpreferably are 2 or more, 3 or more, 4 or more or 5 or more. In oneembodiment, the linker comprises a sequence as described hereinincluding the linker sequences described in the examples such as thesequence GGSGGGGSG (SEQ ID NO: 92).

In one particularly preferred embodiment, a polypeptide incorporatingone or more neo-epitopes such as a polyepitopic polypeptide according tothe present invention is administered to a patient in the form of anucleic acid, preferably RNA such as in vitro transcribed or syntheticRNA, which may be expressed in cells of a patient such as antigenpresenting cells to produce the polypeptide. The present invention alsoenvisions the administration of one or more multiepitopic polypeptideswhich for the purpose of the present invention are comprised by the term“polyepitopic polypeptide”, preferably in the form of a nucleic acid,preferably RNA such as in vitro transcribed or synthetic RNA, which maybe expressed in cells of a patient such as antigen presenting cells toproduce the one or more polypeptides. In the case of an administrationof more than one multiepitopic polypeptide the neo-epitopes provided bythe different multiepitopic polypeptides may be different or partiallyoverlapping. Once present in cells of a patient such as antigenpresenting cells the polypeptide according to the invention is processedto produce the neo-epitopes identified according to the invention.Administration of a vaccine provided according to the inventionpreferably provides MHC class II-presented epitopes that are capable ofeliciting a CD4+ helper T cell response against cells expressingantigens from which the MHC presented epitopes are derived.Administration of a vaccine provided according to the invention may alsoprovide MHC class 1-presented epitopes that are capable of eliciting aCD8+ T cell response against cells expressing antigens from which theMHC presented epitopes are derived. Furthermore, administration of avaccine provided according to the invention may provide one or moreneo-epitopes (including known neo-epitopes and neo-epitopes identifiedaccording to the invention) as well as one or more epitopes notcontaining cancer specific somatic mutations but being expressed bycancer cells and preferably inducing an immune response against cancercells, preferably a cancer specific immune response. In one embodiment,administration of a vaccine provided according to the invention providesneo-epitopes that are MHC class II-presented epitopes and/or are capableof eliciting a CD4+ helper T cell response against cells expressingantigens from which the MHC presented epitopes are derived as well asepitopes not containing cancer-specific somatic mutations that are MHCclass I-presented epitopes and/or are capable of eliciting a CD8+ T cellresponse against cells expressing antigens from which the MHC presentedepitopes are derived. In one embodiment, the epitopes not containingcancer-specific somatic mutations are derived from a tumor antigen. Inone embodiment, the neo-epitopes and epitopes not containingcancer-specific somatic mutations have a synergistic effect in thetreatment of cancer. Preferably, a vaccine provided according to theinvention is useful for polyepitopic stimulation of cytotoxic and/orhelper T cell responses.

The vaccine provided according to the invention may be a recombinantvaccine.

The term “recombinant” in the context of the present invention means“made through genetic engineering”. Preferably, a “recombinant entity”such as a recombinant polypeptide in the context of the presentinvention is not occurring naturally, and preferably is a result of acombination of entities such as amino acid or nucleic acid sequenceswhich are not combined in nature. For example, a recombinant polypeptidein the context of the present invention may contain several amino acidsequences such as neo-epitopes or vaccine sequences derived fromdifferent proteins or different portions of the same protein fusedtogether, e.g., by peptide bonds or appropriate linkers.

The term “naturally occurring” as used herein refers to the fact that anobject can be found in nature. For example, a peptide or nucleic acidthat is present in an organism (including viruses) and can be isolatedfrom a source in nature and which has not been intentionally modified byman in the laboratory is naturally occurring.

Agents, compositions and methods described herein can be used to treat asubject with a disease, e.g., a disease characterized by the presence ofdiseased cells expressing an antigen and presenting a fragment thereof.Particularly preferred diseases are cancer diseases. Agents,compositions and methods described herein may also be used forimmunization or vaccination to prevent a disease described herein.

According to the invention, the term “disease” refers to anypathological state, including cancer diseases, in particular those formsof cancer diseases described herein.

The term “normal” refers to the healthy state or the conditions in ahealthy subject or tissue, i.e., non-pathological conditions, wherein“healthy” preferably means non-cancerous.

“Disease involving cells expressing an antigen” means according to theinvention that expression of the antigen in cells of a diseased tissueor organ is detected. Expression in cells of a diseased tissue or organmay be increased compared to the state in a healthy tissue or organ. Anincrease refers to an increase by at least 10%, in particular at least20%, at least 50%, at least 100%, at least 200%, at least 500%, at least1000%, at least 10000% or even more. In one embodiment, expression isonly found in a diseased tissue, while expression in a healthy tissue isrepressed. According to the invention, diseases involving or beingassociated with cells expressing an antigen include cancer diseases.

According to the invention, the term “tumor” or “tumor disease” refersto an abnormal growth of cells (called neoplastic cells, tumorigenouscells or tumor cells) preferably forming a swelling or lesion. By “tumorcell” is meant an abnormal cell that grows by a rapid, uncontrolledcellular proliferation and continues to grow after the stimuli thatinitiated the new growth cease. Tumors show partial or complete lack ofstructural organization and functional coordination with the normaltissue, and usually form a distinct mass of tissue, which may be eitherbenign, pre-malignant or malignant.

Cancer (medical term: malignant neoplasm) is a class of diseases inwhich a group of cells display uncontrolled growth (division beyond thenormal limits), invasion (intrusion on and destruction of adjacenttissues), and sometimes metastasis (spread to other locations in thebody via lymph or blood). These three malignant properties of cancersdifferentiate them from benign tumors, which are self-limited, and donot invade or metastasize. Most cancers form a tumor but some, likeleukemia, do not. Malignancy, malignant neoplasm, and malignant tumorare essentially synonymous with cancer.

Neoplasm is an abnormal mass of tissue as a result of neoplasia.Neoplasia (new growth in Greek) is the abnormal proliferation of cells.The growth of the cells exceeds, and is uncoordinated with that of thenormal tissues around it. The growth persists in the same excessivemanner even after cessation of the stimuli. It usually causes a lump ortumor. Neoplasms may be benign, pre-malignant or malignant.

“Growth of a tumor” or “tumor growth” according to the invention relatesto the tendency of a tumor to increase its size and/or to the tendencyof tumor cells to proliferate.

For purposes of the present invention, the terms “cancer” and “cancerdisease” are used interchangeably with the terms “tumor” and “tumordisease”.

Cancers are classified by the type of cell that resembles the tumor and,therefore, the tissue presumed to be the origin of the tumor. These arethe histology and the location, respectively.

The term “cancer” according to the invention comprises carcinomas,adenocarcinomas, blastomas, leukemias, seminomas, melanomas, teratomas,lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer,kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skincancer, cancer of the brain, cervical cancer, intestinal cancer, livercancer, colon cancer, stomach cancer, intestine cancer, head and neckcancer, gastrointestinal cancer, lymph node cancer, esophagus cancer,colorectal cancer, pancreas cancer, ear, nose and throat (ENT) cancer,breast cancer, prostate cancer, cancer of the uterus, ovarian cancer andlung cancer and the metastases thereof. Examples thereof are lungcarcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas,renal cell carcinomas, cervical carcinomas, or metastases of the cancertypes or tumors described above. The term cancer according to theinvention also comprises cancer metastases and relapse of cancer.

By “metastasis” is meant the spread of cancer cells from its originalsite to another part of the body. The formation of metastasis is a verycomplex process and depends on detachment of malignant cells from theprimary tumor, invasion of the extracellular matrix, penetration of theendothelial basement membranes to enter the body cavity and vessels, andthen, after being transported by the blood, infiltration of targetorgans. Finally, the growth of a new tumor, i.e. a secondary tumor ormetastatic tumor, at the target site depends on angiogenesis. Tumormetastasis often occurs even after the removal of the primary tumorbecause tumor cells or components may remain and develop metastaticpotential. In one embodiment, the term “metastasis” according to theinvention relates to “distant metastasis” which relates to a metastasiswhich is remote from the primary tumor and the regional lymph nodesystem.

The cells of a secondary or metastatic tumor are like those in theoriginal tumor. This means, for example, that, if ovarian cancermetastasizes to the liver, the secondary tumor is made up of abnormalovarian cells, not of abnormal liver cells. The tumor in the liver isthen called metastatic ovarian cancer, not liver cancer.

The term “circulating tumor cells” or “CTCs” relates to cells that havedetached from a primary tumor or tumor metastases and circulate in thebloodstream. CTCs may constitute seeds for subsequent growth ofadditional tumors (metastasis) in different tissues. Circulating tumorcells arc found in frequencies in the order of 1-10 CTC per mL of wholeblood in patients with metastatic disease. Research methods have beendeveloped to isolate CTC. Several research methods have been describedin the art to isolate CTCs, e.g. techniques which use of the fact thatepithelial cells commonly express the cell adhesion protein EpCAM, whichis absent in normal blood cells. Immunomagnetic bead-based captureinvolves treating blood specimens with antibody to EpCAM that has beenconjugated with magnetic particles, followed by separation of taggedcells in a magnetic field. Isolated cells are then stained with antibodyto another epithelial marker, cytokeratin, as well as a common leukocytemarker CD45, so as to distinguish rare CTCs from contaminating whiteblood cells. This robust and semi-automated approach identifies CTCswith an average yield of approximately 1 CTC/mL and a purity of 0.1%(Allard et al., 2004: Clin Cancer Res 10, 6897-6904). A second methodfor isolating CTCs uses a microfluidic-based CTC capture device whichinvolves flowing whole blood through a chamber embedded with 80,000microposts that have been rendered functional by coating with antibodyto EpCAM. CTCs are then stained with secondary antibodies against eithercytokeratin or tissue specific markers, such as PSA in prostate canceror HER2 in breast cancer and are visualized by automated scanning ofmicroposts in multiple planes along three dimensional coordinates.CTC-chips are able to identifying cytokerating-positive circulatingtumor cells in patients with a median yield of 50 cells/ml and purityranging from 1-80% (Nagrath et al., 2007: Nature 450, 1235-1239).Another possibility for isolating CTCs is using the CellSearch™Circulating Tumor Cell (CTC) Test from Veridex, LLC (Raritan, N.J.)which captures, identifies, and counts CTCs in a tube of blood. TheCellSearch™ system is a U.S. Food and Drug Administration (FDA) approvedmethodology for enumeration of CTC in whole blood which is based on acombination of immunomagnetic labeling and automated digital microscopy.There are other methods for isolating CTCs described in the literatureall of which can be used in conjunction with the present invention.

A relapse or recurrence occurs when a person is affected again by acondition that affected them in the past. For example, if a patient hassuffered from a tumor disease, has received a successful treatment ofsaid disease and again develops said disease said newly developeddisease may be considered as relapse or recurrence. However, accordingto the invention, a relapse or recurrence of a tumor disease may butdoes not necessarily occur al the site of the original tumor disease.Thus, for example, if a patient has suffered from ovarian tumor and hasreceived a successful treatment a relapse or recurrence may be theoccurrence of an ovarian tumor or the occurrence of a tumor at a sitedifferent to ovary. A relapse or recurrence of a tumor also includessituations wherein a tumor occurs at a site different to the site of theoriginal tumor as well as at the site of the original tumor. Preferably,the original tumor for which the patient has received a treatment is aprimary tumor and the tumor at a site different to the site of theoriginal tumor is a secondary or metastatic tumor.

By “treat” is meant to administer a compound or composition as describedherein to a subject in order to prevent or eliminate a disease,including reducing the size of a tumor or the number of tumors in asubject; arrest or slow a disease in a subject; inhibit or slow thedevelopment of a new disease in a subject; decrease the frequency orseverity of symptoms and/or recurrences in a subject who currently hasor who previously has had a disease; and/or prolong, i.e. increase thelifespan of the subject. In particular, the term “treatment of adisease” includes curing, shortening the duration, ameliorating,preventing, slowing down or inhibiting progression or worsening, orpreventing or delaying the onset of a disease or the symptoms thereof.

By “being at risk” is meant a subject, i.e. a patient, that isidentified as having a higher than normal chance of developing adisease, in particular cancer, compared to the general population. Inaddition, a subject who has had, or who currently has, a disease, inparticular cancer, is a subject who has an increased risk for developinga disease, as such a subject may continue to develop a disease. Subjectswho currently have, or who have had, a cancer also have an increasedrisk for cancer metastases.

The term “immunotherapy” relates to a treatment involving activation ofa specific immune reaction. In the context of the present invention,terms such as “protect”, “prevent”, “prophylactic”, “preventive”, or“protective” relate to the prevention or treatment or both of theoccurrence and/or the propagation of a disease in a subject and, inparticular, to minimizing the chance that a subject will develop adisease or to delaying the development of a disease. For example, aperson at risk for a tumor, as described above, would be a candidate fortherapy to prevent a tumor.

A prophylactic administration of an immunotherapy, for example, aprophylactic administration of a vaccine of the invention, preferablyprotects the recipient from the development of a disease. A therapeuticadministration of an immunotherapy, for example, a therapeuticadministration of a vaccine of the invention, may lead to the inhibitionof the progress/growth of the disease. This comprises the decelerationof the progress/growth of the disease, in particular a disruption of theprogression of the disease, which preferably leads to elimination of thedisease.

Immunotherapy may be performed using any of a variety of techniques, inwhich agents provided herein function to remove diseased cells from apatient. Such removal may take place as a result of enhancing orinducing an immune response in a patient specific for an antigen or acell expressing an antigen.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against diseased cells with theadministration of immune response-modifying agents (such as polypeptidesand nucleic acids as provided herein).

The agents and compositions provided herein may be used alone or incombination with conventional therapeutic regimens such as surgery,irradiation, chemotherapy and/or bone marrow transplantation(autologous, syngeneic, allogeneic or unrelated).

The term “immunization” or “vaccination” describes the process oftreating a subject with the purpose of inducing an immune response fortherapeutic or prophylactic reasons.

The term “in vivo” relates to the situation in a subject.

The terms “subject”, “individual”, “organism” or “patient” are usedinterchangeably and relate to vertebrates, preferably mammals. Forexample, mammals in the context of the present invention are humans,non-human primates, domesticated animals such as dogs, cats, sheep,cattle, goats, pigs, horses etc., laboratory animals such as mice, rats,rabbits, guinea pigs, etc. as well as animals in captivity such asanimals of zoos. The term “animal” as used herein also includes humans.The term “subject” may also include a patient, i.e., an animal,preferably a human having a disease, preferably a disease as describedherein.

The term “autologous” is used to describe anything that is derived fromthe same subject. For example, “autologous transplant” refers to atransplant of tissue or organs derived from the same subject. Suchprocedures are advantageous because they overcome the immunologicalbarrier which otherwise results in rejection.

The term “heterologous” is used to describe something consisting ofmultiple different elements. As an example, the transfer of oneindividual's bone marrow into a different individual constitutes aheterologous transplant. A heterologous gene is a gene derived from asource other than the subject.

As part of the composition for an immunization or a vaccination,preferably one or more agents as described herein are administeredtogether with one or more adjuvants for inducing an immune response orfor increasing an immune response. The term “adjuvant” relates tocompounds which prolongs or enhances or accelerates an immune response.The composition of the present invention preferably exerts its effectwithout addition of adjuvants. Still, the composition of the presentapplication may contain any known adjuvant. Adjuvants comprise aheterogeneous group of compounds such as oil emulsions (e.g., Freund'sadjuvants), mineral compounds (such as alum), bacterial products (suchas Bordetella pertussis toxin), liposomes, and immune-stimulatingcomplexes. Examples for adjuvants are monophosphoryl-lipid-A (MPLSmithKline Beecham). Saponins such as QS21 (SmithKline Beecham), DQS21(SmithKline Beecham: WO 96/33739), QS7, QS17, QS18, and QS-L1 (So etal., 1997, Mol. Cells 7: 178-186), incomplete Freund's adjuvants,complete Freund's adjuvants, vitamin E, montanid, alum, CpGoligonucleotides (Krieg et al., 1995, Nature 374: 546-549), and variouswater-in-oil emulsions which are prepared from biologically degradableoils such as squalene and/or tocopherol.

Other substances which stimulate an immune response of the patient mayalso be administered. It is possible, for example, to use cytokines in avaccination, owing to their regulatory properties on lymphocytes. Suchcytokines comprise, for example, inlerleukin-12 (IL-12) which was shownto increase the protective actions of vaccines (cf. Science268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds which enhance an immune response andwhich therefore may be used in a vaccination. Said compounds compriseco-stimulating molecules provided in the form of proteins or nucleicacids such as B7-1 and B7-2 (CD80 and CD86, respectively).

According to the invention, a bodily sample may be a tissue sample,including body fluids, and/or a cellular sample. Such bodily samples maybe obtained in the conventional manner such as by tissue biopsy,including punch biopsy, and by taking blood, bronchial aspirate, sputum,urine, feces or other body fluids. According to the invention, the term“sample” also includes processed samples such as fractions or isolatesof biological samples, e.g. nucleic acid or cell isolates.

The agents such as vaccines and compositions described herein may beadministered via any conventional route, including by injection orinfusion. The administration may be carried out, for example, orally,intravenously, intraperitoneally, intramuscularly, subcutaneously ortransdermally. In one embodiment, administration is carried outintranodally such as by injection into a lymph node. Other forms ofadministration envision the in vitro transaction of antigen presentingcells such as dendritic cells with nucleic acids described hereinfollowed by administration of the antigen presenting cells.

The agents described herein arc administered in effective amounts. An“effective amount” refers to the amount which achieves a desiredreaction or a desired effect alone or together with further doses. Inthe case of treatment of a particular disease or of a particularcondition, the desired reaction preferably relates to inhibition of thecourse of the disease. This comprises slowing down the progress of thedisease and, in particular, interrupting or reversing the progress ofthe disease. The desired reaction in a treatment of a disease or of acondition may also be delay of the onset or a prevention of the onset ofsaid disease or said condition.

An effective amount of an agent described herein will depend on thecondition to be treated, the severeness of the disease, the individualparameters of the patient, including age, physiological condition, sizeand weight, the duration of treatment, the type of an accompanyingtherapy (if present), the specific route of administration and similarfactors. Accordingly, the doses administered of the agents describedherein may depend on various of such parameters. In the case that areaction in a patient is insufficient with an initial dose, higher doses(or effectively higher doses achieved by a different, more localizedroute of administration) may be used.

The pharmaceutical compositions described herein are preferably sterileand contain an effective amount of the therapeutically active substanceto generate the desired reaction or the desired effect.

The pharmaceutical compositions described herein are generallyadministered in pharmaceutically compatible amounts and inpharmaceutically compatible preparation. The term “pharmaceuticallycompatible” refers to a nontoxic material which does not interact withthe action of the active component of the pharmaceutical composition.Preparations of this kind may usually contain salts, buffer substances,preservatives, carriers, supplementing immunity-enhancing substancessuch as adjuvants, e.g. CpG oligonucleotides, cytokines, chemokines,saponin, GM-CSF and/or RNA and, where appropriate, other therapeuticallyactive compounds. When used in medicine, the salts should bepharmaceutically compatible. However, salts which are notpharmaceutically compatible may used for preparing pharmaceuticallycompatible salts and are included in the invention. Pharmacologicallyand pharmaceutically compatible salts of this kind comprise in anon-limiting way those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic acids, and the like. Pharmaceuticallycompatible salts may also be prepared as alkali metal salts or alkalineearth metal salts, such as sodium salts, potassium salts or calciumsalts.

A pharmaceutical composition described herein may comprise apharmaceutically compatible carrier. The term “carrier” refers to anorganic or inorganic component, of a natural or synthetic nature, inwhich the active component is combined in order to facilitateapplication. According to the invention, the term “pharmaceuticallycompatible carrier” includes one or more compatible solid or liquidfillers, diluents or encapsulating substances, which are suitable foradministration to a patient. The components of the pharmaceuticalcomposition of the invention are usually such that no interaction occurswhich substantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions described herein may contain suitablebuffer substances such as acetic acid in a salt, citric acid in a salt,boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may, where appropriate, also containsuitable preservatives such as benzalkonium chloride, chlorobutanol,paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosageform and may be prepared in a manner known per se. Pharmaceuticalcompositions of the invention may be in the form of capsules, tablets,lozenges, solutions, suspensions, syrups, elixirs or in the form of anemulsion, for example.

Compositions suitable for parenteral administration usually comprise asterile aqueous or nonaqueous preparation of the active compound, whichis preferably isotonic to the blood of the recipient. Examples ofcompatible carriers and solvents are Ringer solution and isotonic sodiumchloride solution. In addition, usually sterile, fixed oils are used assolution or suspension medium.

The present invention is described in detail by the figures and examplesbelow, which are used only for illustration purposes and are not meantto be limiting. Owing to the description and the examples, furtherembodiments which are likewise included in the invention are accessibleto the skilled worker.

FIGURES

FIG. 1. Non synonymous cancer-associated mutations are frequentlyimmunogenic and pre-dominantly recognized by CD4⁺ T cells. a, Forimmunogenicity testing, mice (n=5 for b and c, n=3 for d) werevaccinated with either synthetic peptides and poly (I:C) as adjuvant (b)or antigen-encoding RNA (c, d) representing the mutated epitopes (twomutations per mouse). Splenocytes wore restimulated ex vivo with themutated peptide or an irrelevant control peptide and tested by IFNγElispot (sec exemplarily FIG. 2a ) and intracellular cytokine andCD4/CD8 surface staining to assess subtype of elicited immune responses,b-e, T cell responses obtained by vaccinating C57BL/6 mice with epitopesmutated in the B16F10 tumor model. Left, prevalence of non-immunogenic.MHC class I or class II restricted mutated epitopes. Right, examples fordetection and typing of mutation-specific T cells (see Table 1 for dataon individual epitopes). d Left, prevalence of non-immunogenic. MHCclass I or class II restricted mutated epitopes discovered in the CT26model. Right, MHC restriction of immunogenic mutated epitopesprioritized based on predicted MHC class I binding and selected based oneither good (0.1-2.1) or poor (>3.9) binding scores. Sec Table 2 fordata on individual epitopes. Sequences in a (“Sequence analysis andmutation identification”): TTCAGGACCC A (SEQ ID NO: 93);TTCAGGACCCACACGA (SEQ ID NO: 94); TTCAGGACCCACACGACGGGAAGACAA (SEQ IDNO: 95); TTCAGGACCAACACGACGGGAAGACAAGT (SEQ ID NO: 96);CAGGACCCACACGACGGGTAGACAAGT (SEQ ID NO: 97); ACCCACACGACGGGTAG ACAAGT(SEQ ID NO: 98); ACCCACACGAGCCCTAGACAAGT (SEQ ID NO: 99);GACGGGAAGACAAGT (SEQ ID NO: 100). Sequences in b and c: B16-M27 (SEQ IDNO: 10); B16-M30 (SEQ ID NO: 13).

FIG. 2. Efficient tumor control and survival benefit in B16F10 melanomaby immunization with an RNA vaccine encoding a single mutated CD4⁺ Tcell epitope, a, Splenocytes of mice (n=5) vaccinated with B16-M30 RNAwere tested by ELISpot for recognition of synthetic peptides. Left, themutated (B16-M30) versus the corresponding wild type (B16-WT30)sequence. Right, definition of the minimal epitope by testing forrecognition of truncated variants of B16-M30 (mean+SEM), b. The mean+SEMtumor growth (left) and survival (right) of C57BL/6 mice (n=10)inoculated subcutaneously with B16F10 tumors cells and left untreated(control) or immunized IV with B16-M30 encoding RNA (B16-M30) with orwithout administration of CD4 or CD8 depleting antibodies. c, B6 albinomice (n=10) developing lung metastases upon IV injection of luciferasetransgenic B16F10 tumor cells (B16F10-LUC) were treated with B16-M30encoding RNA (B16-M30) or irrelevant control RNA. Median tumor growthwas determined by BLI. d, Single cell suspensions of B16F10 tumors ofuntreated (control, n=x) or B16-M30 RNA immunized mice (n=4) wererestimulated with B16-M30 peptide, medium or irrelevant peptide(VSV-NP52-59) and tested in an IFNγ ELISpot assay (mean+SEM). e. Flowcytometric characterization of tumor infiltrating leucocytes in B16-M30RNA vaccinated mice. Depicted is the frequency of CD4⁺, CD8⁺ orFoxP3⁺/CD4⁺ T cells among CD45⁺ cells and Gr-1⁺/CD11b⁺ cells (MDSCs) ofuntreated (control) or Mut30 RNA vaccinated C57BL/6 mice (n=3)inoculated subcutaneously with B16F10 tumors cells. Sequences in a:B16-M30 (SEQ ID NO: 13): DWENVSPELNSTDQP (SEQ ID NO: 82); DWENVSPELNSTDQ (SEQ ID NO: 81); DWENVSPELNSTD (SEQ ID NO: 82); DWENVSPELNST (SEQ ID NO: 83); DWENVSPELNS (SEQ ID NO: 84); WENVSPELNSTDQP (SEQ IDNO: 85); WENVSPELNSTD (SEQ ID NO: 86); WENVSPELNST (SEQ ID NO: 87);ENVSPELNS TDQP (SEQ ID NO: 88); NVSPELNSTDQP (SEQ ID NO: 89);VSPELNSTDQP (SEQ ID NO: 90).

FIG. 3. Immunization with RNA pen mopes induces T cell responses againstthe individual mutated epitopes and confers disease control andsignificant survival benefit in mouse tumor models. a. Engineering of apoly-neo-epitope RNA vaccine. The RNA pentatope contains five 27mersequences connected by gly/ser linkers inserted into thepST1-Sp-MITD-2hBgUTR-A120 backbone. (UTR, untranslated region; sp.signal peptide; MITD, MHC class I trafficking domain). b, BALB/c mice(n=5) were vaccinated either with pentatope RNA (35 μg) or thecorresponding mixture of five RNA monotopes (7 μg each). T cellresponses in peptide stimulated splenocytes of mice were measured exvivo on day 19 in an IFNγ ELISpot assay (medium control subtractedmean+SEM). c, BALB/c mice (n=10) developing lung metastases upon IVinjection of CT26-LUC cells were treated simultaneously with a mixtureof two RNA pentatopes or left untreated (control). The median tumorgrowth by BLI (left), survival data (mid) and lungs from treated animals(right) are shown. d, CD3 stained tissue sections from the lungs ofpentatope 1+2 treated animals (upper panel). The left side of each panelshows the analyzed sections, the right side the magnifications (scalebar: scan: 1000 μm, upper pictures: 100 μm. lower pictures: 50 μm).CD3⁺, CD4⁺, FoxP3⁺ and CD8⁺ (calculated by CD3⁺ area—CD4⁺ area) areas inconsecutive immunohistochemical lung tissue sections of control (n=6) orRNA pentatope (CD3: n=14; CD4, CD8, FoxP3: n=12) treated animals werequantified and proportions of tumor were calculated. The right figuredepicts a comparison of tumor area in sections of control (n=18) andPentatope1+2 (n=39) treated animals (tumor free animals of pentatope1+2treatment group were excluded). Depicted arc mean±SEM. Sequences in a(“Cloning of template”): GGAAACTTTC (SEQ ID NO: 105).

FIG. 4. RNA pentatope vaccines with mutations selected for in silicopredicted favorable MHC class II binding properties and abundantexpression confer potent antitumor control. a, Comparison of MHC IIbinding scores of immunogenic and non-immunogenic mutations (mediansshown). b, Mutations with high expression levels were selected with(‘ME’ mutations) or without (‘E’ mutations) considering MHC class IIbinding score. See also Table 4. Ten mutations out of each categoryrepresented by two pentatopes each were used for vaccination of CT26-LUClung tumor bearing mice. Tumor growth curves (left), area under thecurve (mid) and ink treated lungs (right) are shown. c, Mice (5 pergroup) were analyzed for T cell responses against the vaccinatedpentatopes by restimulation with RNA electroporated syngeneic BMDC in anIFNγ ELISpot assay. Each dot represents the mean spot count of one mousesubtracted by an irrelevant RNA control (mean±SEM). d, Tumor nodules perlung of BALB/c mice (n=10) inoculated IV with CT26 tumor cells and leftuntreated or injected with irrelevant RNA, pentatope1, pentatope2 orCT26-M19 RNA. e, T cell responses against gp70₄₂₃₋₄₃₁ (gp70-AH1) weredetermined via IFNγ ELISpot assay in blood (pooled from 5 mice, day 20after tumor inoculation) and spleen (n=5). (Background (no peptidecontrol) subtracted mean±SEM depicted). f, Somatic mutation and RNA-Seqdata for individual human cancer samples (black dots) from The CancerGenome Alias (TCGA) was employed to identify genomic (upper panel) andexpressed (mid panel) non-synonymous single nucleotide variations(nsSNVs). (lower panel) Neo-epitopes predicted to bind to the patients'HLA-DRB1 alleles (percentile rank<10%) are shown (SKCM, skin cutaneousmelanoma; COAD, colon adenocarcinoma; BRCA, breast invasive carcinoma).

FIG. 5: Calculation of variant allele frequency (VAF). The figure showsan idealized gene as a combination of exons on a piece of genomic DNA(upper part) and example read sequences aligned to this locus (lowerpart, in a higher zoom level). The site of the mutation event (“mutationsite”) is shown by a dashed line (upper part) or box (lower part). Themutant nucleotides arc colored red, the wild type nucleotides arecolored green. Also the sums of those nucleotides in the VAF formula arecolored accordingly. Sequences in a: TGCAAGAACGCGTACTTATTCGCCGCCATGATTATGACCAGTGTTTCCAGTC (SEQ ID NO: 101); CAAGAACGCGTACTTATTCGCCACCATGATTATGACCAGTGTTTCCAG (SEQ ID NO: 102); AACGCGTACTTATTCGCCACCATGATTATGACCAGTGTTTCCAGTC (SEQ ID NO: 103); TGCAAGAACGCGTACTTATTCGCCGCCATGATTATGACCAGTGTTT (SEQ ID NO: 104).

FIG. 6: Influence of the expression of mutated allele on the predictionperformance of MHC II-scores. 185 selected mutations from the murinetumor models 4T1, CT26 and B16F10 were tested for their antigenicity.The predictive performance of the calculated MHC II-scores was deducedfrom the area under the receiver operating characteristic curve (AUC,open circle). This value was subsequently recalculated after applyingdifferent thresholds for the total mRNA expression (left panel) and theexpression of the mutated allele (right panel, mRNA expression*mutatedallele frequency, closed circles). The maximum AUC values are indicated.The expression of the mutated allele contributes more to the improvementof the prediction performance.

FIG. 7: Comparison of receiver operating characteristic (ROC) curveswith and without threshold on the expression. The ROC curves indicatethe performance of the antigenicity prediction for all 185 selectedmutations from the murine tumor models 4T1, CT26 and B16F10 (dottedcurves) and for those mutations, for which the mRNA expression was ≥6RPKM (left panel, solid curve) or the expression of the mutated allelewas ≥4 RPKM (right panel, solid curve). The selected thresholds achievedthe maximum AUC values (see FIG. 6).

EXAMPLES

The techniques and methods used herein are described herein or carriedout in a manner known per se and as described, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2^(nd) Edition (1989)Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y. Allmethods including the use of kits and reagents are carried out accordingto the manufacturers' information unless specifically indicated.

Example 1: Materials and Methods

Samples. Female 8-12 week old C57BL/6, BALB/c mice (Janvier Labs) andC57BL/6BrdCrHsd-Tyr^(c) mice (B6 albino, Harlan) were kept in accordancewith federal policies on animal research at the University of Mainz.B16F10 melanoma cell line, CT26 colon carcinoma cell line and4T1-luc2-tdtomato (4T1-Luc) cells were purchased in 2010, 2011 and 2011respectively (ATCC CRL-6475 lot #58078645. ATCC CRL-2638 lot #58494154,Caliper 125669 lot #101648) and maintained as suggested by the supplier.Firefly luciferase expressing CT26-Luc and B16F10-Luc cells werelentivirally transduced. Master and working cell banks were generated,of which third and fourth passages were used for tumor experiments.

Next generation sequencing and data processing was described previously(Castle. J. C., et al., Cancer Res 72, 1081 (2012); Castle, J. C, etal., BMC Genomics 15, 190 (2014)). In brief, exome capture from mousetumor cells and tail tissue samples of BALB/c or C57BL/6 mice weresequenced in triplicate (4T1-Luc in duplicate). Oligo(dT) based RNAsequencing libraries for gene expression profiling were prepared intriplicate. Libraries were sequenced on an Illumina HiSeq2000 togenerate 50 nucleotide single-end (BI6F10) or 100 nucleotide paired-end(CT26, 4T1-Luc) reads, respectively. Gene expression values weredetermined by counting reads overlapping transcript exons and junctions,and normalizing to RPKM expression units (Reads which map per kilobaseof transcript length per million mapped reads). Mutation expression wasdetermined by normalization of mutated RNA reads to the total mappedread counts multiplied by 100 million (normalized variant read counts;NVRC).

Mutation selection, validation and prioritization was describedpreviously (Castle, J. C., el al., Cancer Res 72, 1081 (2012); Castle,J. C., et al., BMC Genomics 15, 190 (2014); Lower, M., et al., PLoSComput Biol 8, e1002714 (2012)). Mutations to be pursued were selectedbased on following criteria: (i) present in the respective tumor cellline sequencing triplicates and absent in the corresponding healthytissue sample triplicates, (ii) occur in a RefSeq transcript, and (iii)cause non-synonymous changes. Further criteria were occurrence inexpressed genes of tumor cell lines (median RPKM across replicates). Forvalidation, mutations were amplified from DNA of B16F10, CT26 or 4T1-Luccells and C57BU6 or BALB/c tail tissue and subjected to Sangersequencing. DNA-derived mutations were classified as validated ifconfirmed by either Sanger sequencing or the RNASeq reads. Noconfirmation via Sanger sequencing and immunogenicity testing wasperformed for experiments shown in FIG. 4. For experiments shown in FIG.1 mutated epitopes were prioritized according to their predicted MHCclass I binding based on the consensus method (version 2.5) of theImmune Epitope Database (Vita, R., et al., Nucleic Acids Res 38,D854-D862 (2010)). Mutations targeted in the experiment shown in FIG.4b-e were selected based on either their expression (NVRC) alone ortogether with their predicted MHC class II peptide binding capability(IEDB consensus method version 2.5). Retrospective analysis of MHC IIbinding prediction shown in FIG. 4a was determined with IEDB consensusmethod version 2.12. For analysis of mutations in human tumors. DNAsequencing data of skin cutaneous melanoma (SKCM, n=308). colonadenocarcinoma (COAD, n=192) or breast invasive carcinoma (BRCA, n=872)retrieved from The Cancer Genome Atlas (TCGA) (august 2014) was filteredto obtain genomic non-synonymous point mutations (nsSNVs). RNASeq data(TCGA) of tumor samples with identified genomic mutations was used todefine expressed nsSNVs. In order to predict MHC II binding expressedneo-epitopes seq2HLA was employed to identify the patients' 4-digit HLAclass II (HLA-DQA1, HLA-DQB1, HLA-DRB1) type. The IEDB consensus bindingprediction (version 2.12) was used to predict MHC class II binding froma 27mer peptide and the patients HLA-DRB1 alleles. As recommended fromIEDB, neo-eptiopes with a percentile rank below 10% were considered asbinders.

Synthetic RNA and synthetic peptides. Identified non-synonymousmutations were studied in the context of the respective 27mer amino acidepitope with the mutated amino acid in the center (position 14). Eitherof these mutated peptides were synthesized together with controlpeptides (vesiculo-stomatitis virus nucleo-protein (VSV-NP₅₂₋₅₉),gp70-AH1 (gp70₄₃₂₋₄₃₁) and tyrosinase-related protein 2 (Trp2₁₈₀₋₁₈₈) byJPT Peptide Technologies GmbH. Alternatively, sequences encoding mutated27mer peptides were cloned into the pST1-Sp-MITD-2hBgUTR-A120 backbone(Holtkamp, S., et al,. Blood 108, 4009 (2006)) featuring sequenceelements for pharmacologically optimized synthetic RNA in terms oftranslation efficiency and MHC class I/II processing of epitopes eitheras monotones or as pentatopes fused to each other by sequences encoding10 amino acid long glycine-serine linker in between. Linearization ofthese plasmid constructs, in vitro translation (IVT) of these templatesand purification are described in detail elsewhere (Holtkamp, S., etal., Blood 108, 4009 (2006)).

Mouse Models

For experiments investigating the immunogenicity of mutated epitopesage-matched female C57BL/6 or BALB/c mice were vaccinated on day 0, 3, 7and 14 (immunization with RNA) or day 0 and 7 (immunization withpeptide), the read out was performed five to six days after the lastimmunization. Vaccination was performed cither by retro-orbitalinjection of 200 μl (20 μg per mutation for B16F10, 40 μg per mutationfor CT26) RNA complexed with anionic lipids (manuscript in preparation)or subcutaneous injection of 100 μg synthetic peptide and 50 μg poly(I:C) formulated in PBS (200 μL total volume) into the lateral flank.Two mutations per mouse were tested (n=5 for B16F10, n=3 for CT26). Forconfirmation of immunogenic mutations and subtyping, mice werevaccinated against a single mutation (n=5).

For therapeutic tumor experiments C57BL/6 mice were inoculatedsubcutaneously with 1×10⁵ B16F10 melanoma cells into the right flank andrandomly distributed into treatment groups. Tumor volume was measuredunblinded with a caliper and calculated using the formula (A×B²)/2 (A asthe largest and B the smallest diameter of the tumor). In lungmetastasis experiments 5×10⁵ CT26-Luc or 2×10⁵ CT26 cells were injectedinto the tail vein of BALB/c mice or 1.5×10⁵ B16F10-Luc tumor cells intoB6 albino mice to obtain lung tumors. Tumor growth of luciferasetransgenic cells was traced unblinded by bioluminescence imaging afteri.p. injection of an aqueous solution of D-luciferin (250 μl, 1.6 mg, BDBioscience) on an IVIS Lumina (Caliper Life Sciences). Five minutesafter injection emitted photons were quantified. In vivo bioluminescencein regions of interest (ROI) were quantified as total flux (photons/sec)using IVIS Living Image 4.0 software. Mice were randomized based ontheir total flux values (ANOVA-P method, Daniel's XL Toolbox V6.53).CT26 lung tumor burden was quantified unblinded after tracheal Ink (1:10diluted in PBS) injection and fixation with Fekete's solution (5 mL 10%EtOH, 0.5 mL formalin, and 0.25 mL glacial acetic acid). In therapeuticexperiments mice were administered repealed doses of either monotone (40μg), pentatope RNA (in total 40 μg) or equimolar amounts of irrelevantRNA.

For mechanistic studies repealed doses of CD8 depleting (clone YTS191,BioXcell), CD4 depleting (clone YTS169.1, BioXcell) or CD40L blocking(clone MR1, kind gill of Prof. Stephen Schoenberger) antibodies wereadministered intraperitoneally as indicated in the figure (200 μg/mousein 200 μL PBS).

Enzyme-linked immunospot (ELISpot) has been previously described(Kreiter. S., et al., Cancer Res 70, 9031 (2010)). In brief, 5×10⁵splenocytes were cultured over night at 37° C. in anti-INF-γ (10 μg/mL,clone AN18, Mabtech) coated Multiscreen 96-well plates (Millipore) andcytokine secretion was detected with an anti-IFN-γ antibody (1 μg/mL,clone R4-6A2, Mabtech). For stimulation either 2 μg/mL peptide was addedor spleen cells were coincubated with 5×10⁴ syngeneic bonemarrow-derived dendritic cells (BMDC) transfected with RNA. For analysisof tumor infiltrating lymphocytes, single cell suspensions of lungmetastasis were rested overnight to get rid of living tumor cells viaplastic adherence. Viable cells were separated via density gradientcentrifugation. All retrieved cells were added to the ELISpot plate. Foranalysis of T cell responses in peripheral blood, PBMC were isolated viadensity gradient centrifugation, counted and resimulated by addition ofpeptide and syngeneic BMDC. Subtyping of T cell responses was performedby addition of a MHC class II blocking antibody (20 μg/mL clone M5/114,BioXcell). All samples were tested in duplicates or triplicates.

Flow cytometric analysis was used to determine the subtype of mutationreactive T cells. In the presence of Brefeldin A (Sigma-Aldrich) 2×10⁶splenocytes were stimulated with 2×10⁵ RNA transfected BMDC or 2 μg/mLpeptide. As a positive control splenocytes were treated with phorbol12-myristate 13-acetate (PMA, 0.5 μg/ml, Sigma-Aldrich) and lonomycin (1μg/ml, Sigma-Aldrich). Cells were incubated 5 h at 37° C. andsubsequently stained for CD4⁺ and CD8⁺ cell surface marker. Cells werepermeabilized and fixated using BD Cytofix/Cytoperm according to themanufacturer's protocol and thereafter stained for INF-γ, TNF-α and IL-2cytokines (BD Biosciences). Cytokine secretion among CD4⁺ or CD8⁺ Tcells in stimulated samples was compared to control samples (medium,irrelevant RNA or irrelevant peptide) in order to determine theresponding T cell subtype (n=5). Tumor infiltrating leucocytes wereprepared from subcutaneous B16F10 tumors as described previously (PMID:2071934). The resulting cell suspension was stained for CD4, CD8, Gr-1and CD11b surface marker. Intracellular FoxP3 staining was performedaccording lo the manufacturer's protocol (Mouse Foxp3 Buffer Set, BD).Samples were acquired on a BD FACSCanto II.

Immune histochemistry. Lungs of CT26 tumor bearing mice were fixatedovernight in 4% phosphate buffered formaldehyde solution (Carl Roth) andembedded in paraffin. 50 μm consecutive sections (3 per mouse) werestained for CD3 (clone SP7, Abeam), CD4 (clone 1, cat #50134-M08H, SinoBiologinal) and FoxP3 (polyclonal, cat # NB100-39002, Novus Biologicals)following detection by a HRP-conjugated antibody (Poly-HRP-anti-rabbitIgG, ImmunoLogic) and the corresponding peroxidase substrate (VectorNova Red, Vector Laboratories) and counterstained with hematoxylin.CD3⁺, CD4⁺, FoxP3⁺ and tumor areas were captured on an Axio Scan.Z1(Zeiss) and manually pre-defined tumor and lung regions were quantifiedvia computerized image analysis software (Tissue Studio 3.6.1.Definiens).

Immunofluorescence staining. Cryoconserved organs were cut in 8 μmsections and attached on Superfrost slides. Sections were driedovernight at room temperature (RT) and fixed in 4% para-formaldehyde(PFA) for 10 min at RT in the dark. Sections were washed 3 times withPBS and blocked using PBS supplemented with 1% BSA, 5% mouse serum, 5%rat serum and 0.0275 Nonident for 1 h at RT in the dark. Fluorescentlabeled antibodies (FoxP3, clone FJK-16s, eBioscience; CD8, clone53-6.7, BD; CD4, clone RM4-5, BD) were diluted in staining buffer (PBSsupplemented with 1% BSA, 5% mouse serum and 0.02% Nonident) andsections were stained overnight at 4° C. After washing twice withwashing buffer (PBS supplemented with 1% BSA and 0.02% Nonident) andonce with PBS, slides were stained for 3 min with Hoechst (Sigma),washed 3 times with PBS, once with distilled water and mounted usingMounting Medium Flouromount G (eBioscience). Immunofluorescence imageswere acquired using an epifluorescence microscope (ApoTome, Zeiss).Tumor, CD4, CD8 and FoxP3 stained areas were quantified within manuallypre-defined tumor regions via computerized image analysis software(Tissue Studio 3.6.1., Definiens)

Statistics. Means were compared by using Student's t-test for twogroups. For comparison of means in more than two groups one-way ANOVAwith Tukey's test was applied. The area under the curve (AUC) forcomparison of tumor growth dynamics was determined for single mice pergroup and was displayed as median. Statistical differences in mediansbetween two groups were calculated with a nonparametric Mann-Whitney Utest. Survival benefit was determined with the log-rank test. Allanalyses were two-tailed and carried out using GraphPad Prism 5.03. ns:P>0.05, *: P≤0.05, **: P≤0.01, ***: P≤0.001, Grubb's test was used foridentification of outliers (alpha=0.05).

Example 2: MHC class II Restricted T Cell Epitopes in Neo-EpitopeVaccines A. Characterization or T Cell Subtypes Reactive Against MutatedEpitopes

Recently, we described a workflow for comprehensive mapping ofnon-synonymous mutations of the B16F10 tumor by NGS (FIG. 1a ) (Castle.J. C, et al., Cancer Res 72, 1081 (2012)). Tumor-bearing C57BL/6 micewere immunized with synthetic 27mer peptides encoding the mutatedepitope (mutation in position 14), resulting in T cell responses whichconferred in vivo tumor control. In continuation of that work, we nowcharacterized the T cell responses against the mutated epitopes startingwith those with a high likelihood of MHC I binding. Mice were vaccinatedwith synthetic 27mer mutated epitope peptides (FIG. 1b upper right).Their splenocytes were tested in IFN-γELISpot to identify immunogenicmutations for further analysis of subtype and cytokine expression (FIG.1a ). About 30% of mutated epitopes were found to induce mutationreactive cytokine secreting T cells in mice (FIG. 1b ). Surprisingly,responses against nearly all mutated epitopes (16/17, 95%) were of CD4⁺T cell type (FIG. 1b , Table 1).

TABLE 1Immunogenic B16F10 mutations. B16F10 mutations determined to be immunogenicupon peptide or RNA immunization as described in FIG. 1). (WT, wild type; AA#, number ofmutated amino acid; Mut, Mutation) MHC I Response  Substi- score aftertution Reactive (best vaccination (WT, AA#, T cell predic- with MutationGene Mutated sequence used for vaccination Mut) subtype tion) peptideRNA B16-M05 Eef2 FVVKAYLPVNESFAFTADLRSNTGGQA (SEQ ID NO: 1) G795A CD4⁺1.1 x B16-M08 Ddx23 ANFESGKHKYRQTAMFTATMPPAVERL (SEQ ID NO: 2) V602ACD4⁺ 1.3 x B16-M12 Gnas TPPPEEAMPFEFNGPAQGDHSQPPLQV (SEQ ID NO: 3) S111GCD4⁺ 1.2 x B16-M17 Tnpo3 VVDRNPQFLDPVLAYLMKGLCEKPIAS (SEQ ID NO: 4)G504A CD4⁺ 1.0 x B16-M20 Tubb3FRRKAFLHWYTGEAMDEMEFTEAESNM (SEQ ID NO: 5) G402A CD4⁺ 1.9 x B16-M21Atp11a SSPDEVALVEGVQSLGFTYLRLKDNYM (SEQ ID NO: 6) R552S CD4⁺ 0.1 xB16-M22 Asf1b PKPDFSQLQRNILPSNPRVTRFHINWD (SEQ ID NO: 7) A141P CD4⁺ 1.7x B16-M24 Dag1 TAVITPPTTTTKKARVSTPKPATPSTD (SEQ ID NO: 8) P425A CD4⁺ 2.2x B16-M25 Plod1 STANYNTSHLNNDVWQIFENPVDWKEK (SEQ ID NO: 9) F530V CD4⁺0.1 x x B16-M27 Obsl1 REGVELCPGNKYEMRRHGTTHSLVIHD (SEQ ID NO: 10) T1764MCD8⁺ 2.3 x x B16-M28 Ppp1r7 NIEGIDKLTQLKKPFLVNNKINKIENI (SEQ ID NO: 11)L170P CD4⁺ 3.2 x x B16-M29 Mthfd1lIPSGTTILNCFHDVLSGKLSGGSPGVP (SEQ ID NO: 12) F294V CD4⁺ 1.7 x B16-M30Kif18b PSKPSFQEFVDWENVSPEINSTDQPFL (SEQ ID NO: 13) K739N CD4⁺ 1.2 x xB16-M33 Pbk DSGSPFPAAVILRDALHMARGLKYLHQ (SEQ ID NO: 14) V145D CD8⁺ 0.1 xB16-M36 Tm9sf3 CGTAFFINFIAIYHHASRAIPFGTMVA (SEQ ID NO: 15) Y382H CD4⁺0.2 x B16-M44 Cpsf3l EFKHIKAFDRTFANNPGPMVVFATPGM (SEQ ID NO: 16) D314NCD4⁺ 0.5 x x B16-M45 Mkm1 ECRITSNFVIPSEYWVEEKEEKQKLIQ (SEQ ID NO: 17)N346Y CD4⁺ 1.4 x B16-M46 Actn4NHSGLVTFQAFIDVMSRETTDTDTADQ (SEQ ID NO: 18) F835V CD4⁺ 0.2 x x B16-M47Rpl13a GRGHLLGRLAAIVGKQVLLGRKVVVVR (SEQ ID NO: 19) A24G CD4⁺ 0.5 xB16-M48 Def8 SHCHWNDLAVIPAGVVHNWDFEPRKVS (SEQ ID NO: 20) R255G CD4⁺ 3.8x x B16-M50 Sema3b GFSQPLRRLVLHVVSAAQAERLARAEE (SEQ ID NO: 21) L663VCD4⁺ 2.9 x x

To exclude any bias associated with a peptide-based vaccine format, thisexperiment was repeated using in vitro transcribed (IVT) mRNA encodingthe mutated epitopes (FIG. 1c upper graph right hand side). T cellreactivities determined with these RNA monotopes were largely comparableto the data obtained with synthetic peptides (FIG. 1 c, Table 1), withsomewhat lower numbers of immunogenic epitopes (about 25%). Importantly,also in this setting the majority of mutation-specific immune responses(10/12, ˜80%) were conferred by CD4⁺ T cells.

We extended our study to the chemically induced colon carcinoma modelCT26 (Griswold, D. P. and Corbell, T. H., Cancer 36, 2441 (1975)) inBALB/c mice, in which we recently identified over 1680 non-synonymousmutations (Castle, J. C., et al., BMC Genomics 15, 190 (2014)). Weselected 96 mutations based on their predicted MHC class I bindingproperties. In analogy to the B16F10 study, half of the candidates weregood binders (‘low score’ 0.1-2.1). The other half was deliberatelychosen for poor MHC I binding (‘high score’>3.9). In total, about 20% ofmutated epitopes were immunogenic in mice immunized with the respectiveRNA monotopes (FIG. 1d pie chart, Table 2). It is noteworthy that in the‘low’ MHC I score subgroup a couple of CD8⁺ T cells inducing epitopeswere identified, which was not the case in the “high” score subgroup(FIG. 1d right). This apparently did not bias against MHC class IIrestricted epitopes, as these were represented in similar frequency inboth subgroups constituting the majority of CT26 immunogenic mutations(16/21, 80%).

TABLE 2Immunogenic CT26 mutations. CT26 mutations determined to be immunogenic uponRNA immunization (as described in FIG. 1). (WT, wild type; AA#, number of mutatedamino acid; Mut, Mutation)  MHC I score Reactive (best SubstitutionT cell predic- Mutation Gene Mutated sequence used for vaccination(WT, AA#, Mut) subtype tion) CT26-M03 Slc20a1DKPLRRNNSYTSYIMAICGMPLDSFRA (SEQ ID NO: 22) T425I CD4⁺ 0.3 CT26-M12 Gpc1YRGANLHLEETLAGFWARLLERLFKQL (SEQ ID NO: 23) E165G CD8⁺ 1.9 CT26-M13Nphp3 AGTCCEYWASRALDEEHSIGSMIQLPQ (SEQ ID NO: 24) G234D CD4⁺ 0.1CT26-M19 Tmem87a QAIVRGCSMPGPWRSGRLLVSRRWSVE (SEQ ID NO: 25) G63R CD8⁺0.7 CT26-M20 Slc4a3 PLLPFYPPDEALEIGLELNSSALPPTE (SEQ ID NO: 26) T373ICD4⁺ 0.0 CT26-M24 Cxcr7 MKAFIFKYSAKTGFTKLIDASRVSETE (SEQ ID NO: 27)L340F CD4⁺ 1.6 CT26-M26 E2f8 VILPQAPSGPSYATYLQPAQAQMLTPP (SEQ ID NO: 28)I522T CD8⁺ 0.1 CT26-M27 Agxt2l2EHIHRAGGLFVADAIQVGFGRIGKHFW (SEQ ID NO: 29) E247A CD4⁺ 0.2 CT26-M35Nap114 HTPSSYIETLPKAIKRKINALKQLQVR (SEQ ID NO: 30) V63I CD4⁺ 0.7CT26-M37 Dhx35 EVIQTSKYYMRDVIAIESAWLLELAPH (SEQ ID NO: 31) T646I CD4⁺0.1 CT26-M39 Als2 GYISRVTAGKDSYIALVDKNIMGYIAS (SEQ ID NO: 32) L675I CD8⁺0.2 CT26-M42 Deptor SHDSRKSTSFMSVNPSKEIKIVSAVRR (SEQ ID NO: 33) S253NCD4⁺ 0.3 CT26-M43 Tdg AAYKGHHYPGPGNYFWKCLFMSGLSEY (SEQ ID NO: 34) H169YCD4⁺ 0.3 CT26-M55 Dkk2 EGDPCLRSSDCIDEFCCARHFWTKICK (SEQ ID NO: 35) G192ECD4⁺ 9.7 CT26-M58 Rpap2 CGYPLCQKKLGVISKQFYRISTKTNKV (SEQ ID NO: 36)P113S CD4⁺ 11.3 CT26-M68 Steap2VTSIPSVSNALNWKEFSFIQSTLGYVA (SEQ ID NO: 37) R388K CD4⁺ 6.8 CT26-M75Usp26 KTTLSHTQDSSQSLQSSSDSSKSSRCS (SEQ ID NO: 38) S715L n.d. 5.8CT26-M78 Nbea PAPRAVLTGHDHEIVCVSVCAELGLVI (SEQ ID NO: 39) V576I CD4⁺ 6.3CT26-M90 Aldh18a1 LHSGQNHLKEMAISVLEARACAAAGQS (SEQ ID NO: 40) P154S CD4⁺8.3 CT26-M91 Zc3h14 NCKYDTKCTKADCLPTHMSRRASILTP (SEQ ID NO: 41) P497LCD4⁺ 8.8 CT26-M93 Drosha LRSSLVNNRTQAKIAEELGMQEYAITN (SEQ ID NO: 42)V1189I CD4⁺ 9.9

On a similar note, when analyzing all immune responses to RNA monotonesrepresenting all 38 mutations we identified in the 4T1 mammary carcinomamodel, nearly 70% of the recognized epitopes were recognized by CD4⁺ Tcells (data not shown; Table 3).

TABLE 4Immunogenic 4T1 mutations. 4T1 mutations determined to be immunogenic uponRNA immunization (as described in FIG. 1). (WT, wild type; AA#, number of mutatedamino acid; Mut, Mutation)  Substitution Reactive T cell Mutation GeneMutated sequence used for vaccination (WT, AA#, Mut) subtype 4T1-M2 Gen1IPHNPRVAVKTTNNLVMKNSVCLERDS (SEQ ID NO: 43) K707N CD4 4T1-M3 Polr2aLAAQSLGEPATQITLNTFHYAGVSAKN (SEQ ID NO: 44) M1102I CD4 4T1-M8 Tmtc2QGVTVLAVSAVYDIFVFHRLKMKQILP (SEQ ID NO: 45) V201I CD8 4T1-M14 ZfrAHIRGAKHQKVVTLHTKLGKPIPSTEP (SEQ ID NO: 46) K411T CD4 4T1-M16 Cep120ELAWEIDRKVLHQNRLQRTPIKLQCFA (SEQ ID NO: 47) H66N CD4 4T1-M17 Malt1FLKDRLLEDKKIAVLLDEVAEDMGKCH (SEQ ID NO: 48) T534A CD4 4T1-M20 Wdr11NDEPDLDPVQELIYDLRSQCDAIRVTK (SEQ ID NO: 49) T340I CD8 4T1-M22 Kbtbd2DAAALQMIIAYAYRGNLAVNDSTVEQL (SEQ ID NO: 50) T914 CD4 4T1-M25 Adamts9KDYTAAGFSSFQKLPLDLTSMQIITTD (SEQ ID NO: 51) I623L CD4 4T1-M26 PzpAVKEEDSLHWQRPEDVQKVKALSFYQP (SEQ ID NO: 52) G1199E CD8 4T1-M27 Gprc5aFAICFSCLLAHALNLIKLVRGRKPLSW (SEQ ID NO: 53) F119L CD8 4T1-M30 EnhoMGAAISQGAIIAIVCNGLVGFLL     (SEQ ID NO: 54) L101 CD4 4T1-M31 Dmrta2EKYPKTPKCARCGNHGVVSALKGHKPY (SEQ ID NO: 55) R73G CD4 4T1-M32 RragdSHRSCSHQTSAPSPKALAHNGTPRNAI (SEQ ID NO: 56) L268P CD4 4T1-M35 Zzz3KELLQFKKLKKQNLQQMQAESGFVQHV (SEQ ID NO: 57) K311N CD8 4T1-M39 IlkapRKGEREEMQDAHVSLNDITQECNPPSS (SEQ ID NO: 58) 127S CD4 4T1-M40 CenpfRVEKLQLESELNESRTECITATSQMTA (SEQ ID NO: 59) D1327E CD4

Thus, we have found in three independent mouse tumor models on differentMHC backgrounds that a considerable fraction of non-synonymous cancermutations are immunogenic and that quite unexpectedly the immunogenicmutanome is pre-dominantly recognized by CD4⁺ T cells.

B. MHC Class II Restricted Cancer Mutations as Vaccine Targets

To investigate whether MHC class II restricted cancer mutations are goodvaccine targets in vivo, we proceeded to use synthetic RNA as vaccineformat. Antigen-encoding synthetic RNA is emerging as promising vaccinetechnology due to its advantages including its capability to delivermore than one epitope, its selective uptake by antigen presenting cells(APC) and its intrinsic adjuvanticity (Diken, M., et al., Gene Ther 18,702 (2011); Kreiter. S., et al., Curr Opin Immunol 23, 399 (2011);Pascolo. S., Handb Exp Pharmacol, 221 (2008); Sahin. U., et al., Nat RevDrug Discov 13, 759 (2014); Van, L. S., et al., Hum Vaccin Immunother 9(2013)). Our group has developed pharmacologically optimized RNA(stabilizing elements in RNA sequence and liposomal formulation), whichmeanwhile has reached the stage of clinical testing (NCT01684241)(Holtkamp, S., et al., Blood 108, 4009 (2006); Kreiter. S., et al., JImmunol 180, 309 (2008); Kuhn. A. N., et al. Gene Ther 17, 961 (2010)).We engineered RNA encoding B16-M30, one of the epitopes identified inthe B16F10 tumor model. B16-M30 elicited strong CD4⁺ T cell responses,which did not recognize the wild type peptide (FIG. 2a left) as themutated amino acid was shown to be essential for T cell recognition(FIG. 2a right). When B16F10 tumor-bearing C57B176 mice were repeatedlyvaccinated with the B16-Mt30 RNA monotope, tumor growth was profoundlyretarded (FIG. 2b ). Half of the B16-M30 RNA treated mice were stillalive 120 days after tumor vaccination, while all the control RNAtreated mice died within 65 days.

Similarly, repeated vaccination in a lung metastasis model withluciferase transduced B16F10 cells revealed efficient eradication ofmetastases with B16-M30 RNA but not control synthetic RNA in the vastmajority of mice as shown by bioluminescence imaging (BLI) (FIG. 2c ).Consistently, tumor infiltrating leukocytes purified from B16F10 tumorsof B16-M30 RNA immunized mice showed strong reactivity against B16-M30(FIG. 2d ).

Taking together, these data establish B16-M30 as a novel major rejectionantigen in B16F10 tumors. They also exemplify that immunizing with RNAencoding a single immunogenic mutated epitope may give rise tofunctional T cells. These cells appear to be capable to target into thecancer lesion triggering control and even cure in murine tumor models.Our findings are in agreement with recent reports supporting the pivotalrole of CD4⁺ T cell immunity in the control of cancer (Schumacher, T.,et al., Nature 512, 324 (2014); Tran, E., et al., Science 344, 641(2014)).

As the vast majority of mutations are unique to the individual patient,tapping the mutanome as a source for vaccine antigens requires anactively individualized approach (Britten, C. M., et al., Nat Biotechnol31, 880 (2013)). In this respect, one of the major challenges is instantmanufacturing of a tailored on-demand vaccine. This can be viablyaddressed by RNA vaccine technology. RNA manufacturing based on in vitrotranscription usually takes a few days (FIG. 3a ). At present, theGMP-grade material could be made ready for release within three weeksand this process is continuously being optimized to reduce the duration.On another note, though we have shown tumor eradication in mouse modelswith a single mutation, one would ideally prefer to combine severalmutations in a poly-neo-epitope vaccine. This would allow us to addressseveral factors that counteract the clinical success of vaccines inhumans such as tumor heterogeneity and immunoediting (Gerlinger, M., etal., N Engl J Med 366, 883 12012); Koebel, C. M., et al., Nature 450,903 (2007)).

In light of these considerations, we explored how to use our insights onimmunogenic epitopes to develop a cancer vaccine concept which we call“mutanome engineered RNA immunotherapy” (MERIT) (FIG. 3a ). To test thisconcept, we selected four MHC class II (CT26-M03, CT26-M20, CT26-M27,CT26-M68) and one MHC class I (CT26-M19) restricted mutations that werederived from the CT26 model (sec Table 2) and engineered RNA monotopesencoding each of them. In addition, a synthetic RNA pentatope wasengineered encoding all five mutated epitopes connected by 10mernon-immunogenic glycine/serine linkers to avoid the generation ofjunctional epitopes (FIG. 3a ). By immunizing naïve BALB/c mice we foundthat the quantity of IFN-producing T cells elicited by the pentatope wascomparable to that evoked by the respective monotone for three of thesemutations (FIG. 3b ). However, for two of these mutations the pentatopeRNA was significantly superior in robustly expanding mutations-specificT cells.

We assessed the anti-tumour efficacy of immune responses elicited by RNApentatope vaccines in a lung metastasis model of CT26 luciferasetransfectant (CT26-Luc) tumors. Tumor-bearing BALB/c mice werevaccinated repeatedly with a mixture of two RNA pentatopes (3 MHC classI and 7 class II restricted epitopes) including the mutations tested inthe previous experiment. Tumor growth in vaccinated mice wassignificantly inhibited as measured by BLI of the lung (FIG. 3c left).At day 32 all mice in the RNA pentatope group were alive whereas 80% ofthe control mice had already died (FIG. 3c mid). Post mortem macroscopic(FIG. 3c right), histological (FIG. 3d right) and computerized imageanalysis (data not shown) of tissue sections revealed significantlylower tumor load in the vaccinated mice as compared to untreatedcontrols. Tumor lesions of pentatope RNA vaccinated mice were brisklyinfiltrated with CD3⁺ T cells, whereas the number of CD3⁺ T cells wassignificantly lower in their surrounding lung tissues. Tumors ofuntreated controls displayed CD3⁺ cells staining which was not muchdifferent to that of the surrounding lung tissue in terms of quantityand mainly at the tumor border but not within the tumor. (FIG. 3d ).

Altogether, these findings indicate that T cells against each singleepitope are elicited with a MERIT approach employing a poly-neo-epitopeencoding RNA vaccine. These T cells target tumor lesions, recognizetheir mutated targets and result in efficient tumor control in vivo.

C. Selection of Mutations Having Anti-Tumor Immunity

One of the key questions is how to select the mutations with the highestprobability of inducing efficient anti-tumor immunity. We (FIG. 1dright) and others (Matsushita, H., et al., Nature 482, 400 (2012);Robbins. P. F., et al., Nat Med 19, 747 (2013); van, R. N., el al., JClin Oncol 31, e439-e442 (2013)) have shown that MHC class I bindingscores enable enrichment for mutated epitope candidates which elicitCD8⁺ responses and tumor rejection (Duan, F., et al., J Exp Med 211,2231 (2014)). Our findings described above indicate that MHC class IIpresented mutated epitopes may even be of higher interest for a MERITapproach. In fact, a correlation analysis revealed that immunogenicmutations have a significantly belter MHC class II binding score ascompared to non-immunogenic ones (FIG. 4a ). Most cancers lack MHC classII expression. Effective recognition of neo-epitopes by CD4⁺ T cells inMHC class II negative tumors should depend on release of tumor antigensto be taken up and presented by antigen presenting cells (APCs). Thisshould be most efficient for antigens with highly abundant expression.To test this hypothesis, we implemented an algorithm combining good MHCclass II binding with abundant expression of the mRNA encoding themutated epitope. For the latter we used confirmed mutated RNA sequencingreads normalized to the overall read count (NVRC: normalized variantread counts). We ranked CT26 mutanome data with this algorithm andselected the top ten mutations (‘ME’ mutations in Table 4) predicted tobe good MHC class II binders among the most abundant candidate epitopes(NVRC≥60). As control we chose ten mutations based on abundantexpression only (‘E’ mutations in Table 4). Most importantly, theseepitopes were used without any further pre-validation or immunogenicitytesting to engineer two RNA pentatopes for each group (P_(ME) and P_(E)pentatopes). When mice with established CT26-Luc lung tumors werevaccinated with these epitopes, P_(ME) as compared to P_(E) pentatopesinduced a much stronger T cell response (FIG. 4c ). Established lungmetastases were completely rejected in almost all mice whereas P_(E)pentatopes were not able to confer tumor growth control (FIG. 4b ).

TABLE 4In silico prediction of CT26 mutations with abundant expression and favorableMHC class II binding properties. CT26 mutations selected for high expression with (ME) orwithout (E) consideration of the MCH II percentile rank (IEDB consensus version 2.5). (WT,wild type; AA#, number of mutated amino acid; Mut, Mutation ) MHC IIscore (best Substitution Expression predic- Mutation GeneMutated sequence used for vaccination (WT, AA#, Mut) (NVRC) tion)CT26-E1 Asns DSVVIFSGEGSDEFTQGYIYFHKAPSP (SEQ ID NO: 60) L370F 1428.0545.45 CT26-E2 Cd24 PQTSPTGILPTTSNSISTSEMTWKSSL (SEQ ID NO: 61) D120N1150.85 23.76 CT26-E3 Actb WIGGSILASLSTFHQMWISKQEYDESG (SEQ ID NO: 62)Q353H 974.16 8.30 CT26-E4 Tmbim8SALGSLALMIWLMTTPHSHETEQKRLG (SEQ ID NO: 63) A73T 825.51 2.96 CT26-E5Glud1 DLRTAAYVNAIEKIFKVYNEAGVTFT  (SEQ ID NO: 64) V546I 619.54 8.01CT26-E16 Eif4g2 KLCLELLNVGVESNLILKGVILLIVDK (SEQ ID NO: 65) K108N 327.7920.99 CT26-E17 Sept7 NVHYENYRSRKLATVTYNGVDNNKNKG (SEQ ID NO: 66) A314T316.98 6.47 CT26-E18 Fn1 YTVSVVALHDDMENQPLIGIQSTAIPA (SEQ ID NO: 67)S1710N 303.62 17.41 CT26-E19 Brd2KPSTLRELERYVLACLRKKPRKPYTIR (SEQ ID NO: 68) S703A 301.83 7.86 CT26-E20Uchl3 KFMERDPDELRFNTIALSAA        (SEQ ID NO: 69) A224T 301.78 9.75CT26-M1 Aldh18a1 LHSGQNHLKEMAISVLEARACAAAGQS (SEQ ID NO: 70) P154S 67.730.05 CT26-M2 Ubqin1 DTLSAMSNPRAMQVLLQTQQGLQTLAT (SEQ ID NO: 71) A62V84.08 0.24 CT26-M3 Ppp6r1 DGQLELLAQGALDNALSSMGALHALRP (SEQ ID NO: 72)D309N 139.80 0.44 CT26-M4 Trip12WKGGPVKIDPLALMQAIERYLVVRGYG (SEQ ID NO: 73) V1328M 83.09 0.49 CT26-M5Pcdhgc3 QDINDNNPSFPTGKMKLEISEALAPGT (SEQ ID NO: 74) E139K 86.16 0.54CT26-M6 Cad SDPRAAYFRQAENFMYIRMALLATVLG (SEQ ID NO: 75) G2139D 152.860.55 CT26-M7 Smarcd1 MDLLAFERKLDQTVMRKRLDIQEALKP (SEQ ID NO: 76) I161V125.85 0.60 CT26-M8 Ddx27 ITTCLAVGGLDVKFQEAALRAAPDILI (SEQ ID NO: 77)S297F 61.82 0.62 CT26-M9 Snx5KARLKSKDVKLAEAHQQECCQKFEQLS (SEQ ID NO: 78) T341A 120.27 0.73 CT26-M10Lin7c GEVPPQKLQALQRALQSEFCNAVREVY (SEQ ID NO: 79) V41A 71.24 1.09

Antigen specific T_(H) cells promote the cross-priming of tumor specificCTL responses by CD40 ligand mediated licensing of dendritic cells. Thismay result in antigen spread if T_(H) cells recognize their antigen onthe same APC that cross-presents an unrelated CTL epitope (Bennett, S.R., et al., Nature 393, 478 (1998); Schoenherger. S. P., et al., Nature393, 480 (1998)). Congruently, in the blood and spleen of mice immunizedwith P_(ME) but not P_(E) pentatopes we detected strong CD8⁺ T cellresponses against gp70-AH1, a well characterized immunodominant CTLepitope derived from the endogenous murine leukemia virus-related cellsurface antigen (FIG. 4d ). This indicates that cancer neo-epitopespecific T_(H) cells, in analogy to viral neo-antigen specific T cells(Croxford, J. L., et al., Autoimmun Rev 1, 251 (2002)), may exert theiranti-tumour function by antigen spreading and augmentation of CTLresponses

D. Summary

In summary, our data indicate that MHC class II restricted T cellepitopes are abundant in the cancer mutanome and can be used tocustomize RNA-based poly-neo-epitope vaccines with substantialtherapeutic effect in mouse tumour models.

The mechanism responsible for the high rate of CD4⁺ T cell recognitionof mutations is unclear yet. A simple explanation may be the longer andvariable size of peptides presented on MHC class II molecules ascompared to MHC class I epitopes increasing the likelihood that amutation is covered by the respective peptide. T cell epitopes presentedby MHC class I molecules are typically peptides between 8 and 11 aminoacids in length with well-defined N- and C-termini. MHC class IImolecules present longer peptides of 13-17 amino acids in length with a9 amino acid MHC II core binding region and variable number ofadditional flanking amino acids both contributing to the recognition byCD4⁺ T cells (Arnold, P. Y., et al., J Immunol 169, 739 (2002)).

While the first evidence of the spontaneous CD8⁺ and CD4⁺ T-cellresponses directed against mutated gene-products in cancer patients wasgenerated in the 1990s (Dubey, P., et al., J Exp Med 185, 695 (1997);Lennerz, V., et al., Proc Natl Acad Sci USA 102, 16013 (2005); Wolfel,T., et al., Science 269, 1281 (1995)), only the recent high levelpublications have created broad acceptance for the enormous potential ofmutation-specific T cells to confer anti-tumor activity in cancerpatients (Lu, Y. C., et al., J Immunol 190, 6034 (2013); Schumacher, T.,et al., Nature 512, 324 (2014); Tran, E., et al., Science 344, 641(2014)). To assess whether the principles we unraveled in the mousemodels for melanoma, colon and breast cancer are true in the humansetting, we analyzed mutation and RNA-Seq data in the same three humancancer types provided by The Cancer Genome Atlas (TCGA). For all threehuman cancer types we confirmed the abundance of mutations predicted tobind to MHC class II we revealed in mouse models (FIG. 4.e).

The MERIT approach we presented here integrates advances in the field ofnext generation sequencing, computational immunology and syntheticgenomics and thereby provides the integrated technology forcomprehensive exploitation of the neo-epitope target repertoire.Targeting multiple mutations at once may at least in theory pave the wayto solve critical problems in current cancer drug development such asclonal heterogeneity and antigen escape (Kroemer, G. and Zitvogel, L.,Oncoimmunology 1, 579 (2012); Mittal, D., et al., Curr Opin Immunol 27,16 (2014)).

Meanwhile, based on this study and our prior work clinical translationhas been initiated and a first-in-concept trial in melanoma patients(Castle, J. C., el al., Cancer Res 72, 1081 (2012); Castle, J. C., etal., Sci Rep 4, 4743 (2014); Lower, M., et al., PLoS Comput Biol 8,e1002714 (2012)) is actively recruiting (NCT02035956) and confirms that“just in time” production of a poly-neo-epitope mRNA cancer vaccine isin fact feasible.

Example 3: Selection of Mutations Having Anti-Tumor Immunity

For selecting/ranking amino acid sequence modifications one may proceedas follows:

-   -   1. Within a given list of non-synonymous point mutations,        compute a peptide sequence which has the mutated amino acid in        the middle and is flanked by up to 13 amino acids on the N and        C-terminal end, respectively; this will be called 27mer in the        following text (the length for each flanking sequence may be        smaller than 13 amino acids when the mutation is close to the N        or C-terminus of the whole protein)    -   2. Compute MHC class II binding prediction consensus scores        (e.g. using the IEDB T-cell prediction tools [Wang P. et        al. (2010) BMC Bioinformatics. 11:568. PMID: 21092157.        http://tools.immuneepitope.org/mhcii/]) for each overlapping 15        nt long subsequence of each 27mer; the best (=lowest) score is        assigned to the whole 27mer    -   3. Compute the expression (preferably in RPKM units [Ali        Mortazavi, et al. (2008) Nature Methods 5, 621-628]) of the        genes to which the 27mers are associated    -   4. Compute the variant allele frequency (VAF) of each mutation        in the RNA:        -   input are short read alignments of an RNA-Seq experiment            done with the same tumor sample as used for mutation            detection        -   look up the alignments and reads overlapping the mutation            site        -   tally the nucleotides mapped to the mutation site using the            reads aggregated a step before        -   compute the sum of mutant-allele nucleotides divided by the            sum of all nucleotides mapped to the genomic site of the            mutation (FIG. 5)    -   5. Multiply the respective gene expression with the VAF to get        the mutation expression (preferably in RPKM units)    -   6. Rank all 27mers by the MHC binding score (as computed in step        2, lowest score is best) and remove 27mers with an associated        mutation expression of less than a given threshold

Application to Murine Data Set:

For testing the algorithm, 185 mutations were selected from the murinetumor models 4T1, CT26 and B16F10 were tested for their antigenicity.Then we first tried to lest the influence of the level of gene andmutation expression on the predictive performance of the algorithm (FIG.6). Here we can observe that the maximum area under the curve of thereceiver operating characteristic (ROC AUC [Fawcett T., Pattern RecognLett. 2006; 27:861-874. doi: 10.1016/j.patree.2005.10.010]) is higherwhen the mutation expression is filtered instead of the gene expression(FIG. 6 left (gene expression) vs. right (mutation expression) plot).FIG. 7 shows the ROC curves for the optimum thresholds, indicating apronounced influence of the mutation expression for binders with only amediocre relative binding affinity (FIG. 7, right panel, values betweena false positive rate of about 0.3 and 0.6).

1. A method for predicting immunogenic amino acid modifications, themethod comprising the steps: a) ascertaining a score for binding of amodified peptide which is a fragment of a modified protein to one ormore MHC class II molecules, wherein the modified peptide comprises oneor more amino acid modification. and b) ascertaining a score forexpression or abundance of the modified protein.
 2. The method of claim1, wherein a score for binding to one or more MHC class II moleculesindicating binding to one or more MHC class II molecules and a score forexpression or abundance of the modified protein indicating expression,high level of expression or abundance of the modified protein indicatesthat the modification or modified peptide is immunogenic.
 3. A methodfor selecting and/or ranking immunogenic amino acid modifications, themethod comprising the steps: a) ascertaining a score for binding of amodified peptide which is a fragment of a modified protein to one ormore MHC class II molecules. and b) ascertaining a score for expressionor abundance of the modified protein, wherein the method comprisesperforming steps a) and b) on two or more different modifications. 4.The method of claim 3, wherein the different modifications are presentin the same and/or in different proteins.
 5. The method of claim 3 whichcomprises comparing the scores of said two or more differentmodifications.
 6. The method of claim 3, wherein the scores of said twoor more different modifications are compared by ranking the differentmodifications by their MHC class II binding scores and removingmodifications with an expression or abundance of less than a giventhreshold.
 7. The method of claim 1, wherein the score for binding toone or more MHC class II molecules reflects a probability for binding toone or more MHC class II molecules.
 8. The method of claim 1 whichcomprises performing step a) on two or more different modified peptides,said two or more different modified peptides comprising the same one ormore modifications.
 9. The method of claim 8, wherein the two or moredifferent modified peptides comprising the same one or more modificationcomprise different fragments of a modified protein, said differentfragments comprising the same one or more modification present in theprotein.
 10. The method of claim 8, wherein the two or more differentmodified peptides comprising the same one or more modification comprisedifferent potential MHC class II binding fragments of a modifiedprotein, said fragments comprising the same one or more modificationpresent in the protein.
 11. The method of claim 8 further comprisingselecting (the) modified peptide(s) from the two or more differentmodified peptides comprising the same one or more modifications having aprobability or having the highest probability for binding to one or moreMHC class II molecules.
 12. The method of claim 8, wherein the two ormore different modified peptides comprising the same one or moremodifications differ in length and/or position of the one or moremodifications.
 13. The method of claim 8, wherein the best score forbinding to one or more MHC class II molecules of the two or moredifferent modified peptides comprising the same one or moremodifications is assigned to the modifications.
 14. The method of claim1, wherein ascertaining a score for expression or abundance of amodified protein comprises determining the level of expression of theprotein to which the modification is associated and determining thefrequency of the modified protein among the protein to which themodification is associated.
 15. The method of claim 14, wherein saiddetermining the level of expression of the protein to which themodification is associated and/or determining the frequency of themodified protein among the protein to which the modification isassociated is performed on the RNA level.
 16. The method of claim 14,wherein the frequency of the modified protein among the protein to whichthe modification is associated is determined by determining the variantallele frequency.
 17. The method of claim 16, wherein the variant allelefrequency is the sum of detected sequences, in particular reads,covering the mutation site and carrying the mutation divided by the sumof all detected sequences, in particular reads, covering the mutationsite.
 18. The method of claim 1, wherein for ascertaining a score forexpression or abundance of a modified protein a score for the level ofexpression of the protein to which the modification is associated ismultiplied with a score for the frequency of the modified protein amongthe protein to which the modification is associated.
 19. The method ofclaim 1, wherein the modified peptide comprises a fragment of themodified protein, said fragment comprising the modification present inthe protein.
 20. The method of claim 1 further comprising identifyingnon-synonymous mutations in one or more protein-coding regions.
 21. Themethod of claim 1, wherein amino acid modifications are identified bypartially or completely sequencing the genome or transcriptome of one ormore cells such as one or more cancer cells and optionally one or morenon-cancerous cells and identifying mutations in one or moreprotein-coding regions.
 22. The method of claim 20, wherein saidmutations are somatic mutations.
 23. The method of claim 20, whereinsaid mutations are cancer mutations.
 24. (canceled)
 25. (canceled)
 26. Amethod for providing a vaccine comprising the step: identifying one ormore modifications or one or more modified peptide predicted asimmunogenic or more immunogenic by the method of claim 1; and producinga vaccine comprising a peptide or polypeptide comprising the one or moremodification or one or more modified peptide predicted as immunogenic ormore immunogenic, or a nucleic acid encoding the peptide or polypeptide.27. (canceled)
 28. A vaccine produced according to the method of claim26.